同位素化學標記法搭配質譜技術進行發炎反應動物模式之差異蛋白質體學研究

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(1)國立臺灣師範大學化學系研究所博士論文 Department of Chemistry, National Taiwan Normal University Doctoral Dissertation. 同位素化學標記法搭配質譜技術進行發炎反應動物模式之差異蛋白質體學研究 Differential Proteomics of Monosodium Urate Crystals-Induced Responses in Dissected Murine Air Pouch Membranes by iTRAQ Technology. 邱芷葳. 撰. Chih-Wei Chiu 指導教授：陳頌方博士 Advisor: Sung-Fang Chen, Ph.D. 中華民國一百零四年七月 July, 2015.

(2) 謝誌經過漫長的六年，博士班的生涯在此劃下句點，我也跟著 C404 實驗室漸漸的成長茁壯。指導教授陳頌方老師總是不辭辛勞的悉心引導，給予很多學習的機會。除了在專業知識的啟發與傳授之外，小至日常生活的禮儀、待人處事原則，大至社會的禮教法規老師都不厭其煩的叮囑教導。在此，對老師深切表達由衷的謝意。謝謝老師，您辛苦了! 感謝我的同學們，德蕙、育廷、昀諭。當初一起努力設置實驗室，從空蕩蕩的教室，到如今令人羨慕的專業質譜實驗室，當年我們都只是懵懂無知小毛頭們，和你們互相討論，互相打氣，是研究所生涯中最懷念的時光。沛倫學長，謝謝您對我的照顧，總是像大哥哥一樣在一旁關心著實驗室的大家。子瑋、俞靜、郡倫，謝謝你們包容我的壞脾氣，並且教我怎麼當一個好學姐。立平、珮琳、群皓、堯聰，謝謝你們陪我度過艱困的時光。祖儀、佳穎、昀昇、羽薇，謝謝你們帶給我歡笑與許多的加油鼓勵。思樺、瓊文、境晏，謝謝你們在我焦頭爛額時對我伸出援手，幫了我好多忙。婷婷、詩潔、怡安、廷宇，謝謝你們無限的正面能量支援，讓我最後一年的研究所生活充滿活力。另外，還要特別感謝陳柏睿經理，雖然工作繁忙但仍守護著 C404 的每一台質譜儀，而且總是對我有問必答，讓我對質譜儀有更深入的了解。謝謝黃興鴻學長，教導我許多專業知識與珍貴的經驗分享。謝謝曾鈺秀小姐教導我關於 HPLC 的知識，平時也總是對我關懷有加。感謝何國榮老師、周綠蘋老師、林震煌老師在百忙之中抽空前來擔任我的口試委員，並不吝惜對本論文給予指導與建議，使內容更趨完善。謝謝陳翰民老師在實驗上的慷慨協助、細心指導，才讓論文最後能夠順利的發表。最後，感謝我的家人、我的摯友 Aidan、Jackie、Lilico、Stacy、QQ，謝謝你們對我的體諒、支持與鼓勵，讓我能順利完成我的博士學位。僅將此本論文獻給一路上幫助我的人，以表達我無限的感激。.

(3) Table of Content Table of Content ............................................................................................ i List of Tables ................................................................................................ iv List of Figures ............................................................................................... v ABBREVIATIONS ......................................................................................... vii ABSTRACT .................................................................................................... x INTRODUCTION ............................................................................................ 1 Peptide Fractionation ................................................................................................... 1 Differential Proteomics ................................................................................................ 5 Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) ........................................ 6 Pulsed-Q Dissociation (PQD)......................................................................................... 6 Monosodium Urate Crystals-Induced Responses in Dissected Murine Air Pouch Membranes ................................................................................................................. 7. EXPERIMENTAL SECTION ............................................................................ 11 Chemicals .................................................................................................................. 11 Sample Preparation and Digestion of PLC/PRF/5 lysate ............................................... 11 Protein Standard Mixture Preparation and iTRAQ Reagent Labeling for LTQ-PQD optimization .............................................................................................................. 12 Murine Air Pouches.................................................................................................... 13 Dissection of the Air Pouch Membrane ....................................................................... 14 Air Pouch Membrane Protein Extraction ..................................................................... 14 i.

(4) Murine Air Pouch Sample Pretreatment Preparation and iTRAQ Reagent Labeling ....... 15 Analytical Setups for Peptide from the PLC/PRF/5 Lysate Pretreatment ....................... 16 Analytical Setups for First-Dimension Separation for the PLC/PRF/5 Lysate Sample ...... 16 SCX Chromatography.................................................................................................. 17 HILIC Chromatography ............................................................................................... 18 Reverse Phase Chromatography at Low pH ................................................................. 18 Reverse Phase Chromatography at High pH ................................................................. 19 Solution-IEF Separation .............................................................................................. 20 LC-MS/MS Analysis .................................................................................................... 21 LC-MS/MS Analysis by LTQ-PQD ................................................................................. 22 Peptide and Protein Identification and Quantitation ................................................... 23 Western Blotting ........................................................................................................ 25 Treatment of THP-1 Cells with a Suspension of MSU Crystals ....................................... 25 Total RNA Isolation, Reverse Transcription, Real Time PCR Quantification of Genes ...... 26. RESULTS AND DISCUSSION ......................................................................... 27 Part I - Evaluation of Peptide Fractionation Strategies used in Proteome Analysis. ....... 27 The Influence of Salt on Separation Efficiency ......................................................... 28 Orthogonality of Two-Dimensional Separations ...................................................... 29 Charge, GRAVY and pI Value Distribution of Peptides .............................................. 31 Complementarity of SCX x RPLC, HILIC x RPLC, Alkaline-RP x RPLC and sIEF x RPLC Methods ................................................................................................................ 33 Summary ............................................................................................................... 36. ii.

(5) PART II - Optimization of Pulsed-Q Dissociation Parameters in Linear Ion Trap Mass Spectrometer for iTRAQ Quantitation ......................................................................... 37 Effects of Microscan and Target Value ..................................................................... 37 Effects of Normalized Collisional Energy.................................................................. 38 Effects of Activation Q and Delay Time ................................................................... 39 Summary ............................................................................................................... 41 PART III - Differential Proteomics of Monosodium Urate Crystals-Induced Responses in Dissected Murine Air Pouch Membranes by iTRAQ Technology.................................... 42 iTRAQ Proteomics Profiling of MSU Crystals in the Air Pouch Membrane by 2D LC-MS/MS Analysis ................................................................................................ 43 MSU Crystals Stimulated the Alternative Pathway of the Complement System......... 45 Up-Regulated Proteins Related to NALP3 Inflammasome ........................................ 47 The TCA Cycle Is Down-Regulated at Transcription and Translation Level by MSU-Stimulated Inflammation ............................................................................... 48 Summary ............................................................................................................... 53. CONCLUSIONS ............................................................................................ 54 REFERENCES ............................................................................................... 56. iii.

(6) List of Tables Table 1 ........................................................................................................ 64 Table 2 ........................................................................................................ 65 Table 3 ........................................................................................................ 66 Table 4 ........................................................................................................ 67 Table 5 ........................................................................................................ 68 Table 6 ........................................................................................................ 69 Table 7 ........................................................................................................ 70 Table 8 ........................................................................................................ 71. iv.

(7) List of Figures Figure 1 ...................................................................................................... 88 Figure 2 ...................................................................................................... 89 Figure 3 ...................................................................................................... 90 Figure 4 ...................................................................................................... 91 Figure 5 ...................................................................................................... 94 Figure 6 ...................................................................................................... 95 Figure 7 ...................................................................................................... 96 Figure 8 ...................................................................................................... 97 Figure 9 ...................................................................................................... 98 Figure 10 .................................................................................................... 99 Figure 11 .................................................................................................. 100 Figure 12 .................................................................................................. 101 Figure 13 .................................................................................................. 102 Figure 14 .................................................................................................. 103 Figure 15 .................................................................................................. 104 Figure 16 .................................................................................................. 105 Figure 17 .................................................................................................. 106 Figure 18 .................................................................................................. 107 Figure 19 .................................................................................................. 109 Figure 20 .................................................................................................. 110 Figure 21 .................................................................................................. 111 Figure 22 .................................................................................................. 112 Figure 23 .................................................................................................. 113 Figure 24 .................................................................................................. 114 v.

(8) Figure 25 .................................................................................................. 115 Figure 26 .................................................................................................. 116 Figure 27 .................................................................................................. 117 Figure 28 .................................................................................................. 118 Figure 29 .................................................................................................. 119 Figure 30 .................................................................................................. 120 Figure 31 .................................................................................................. 121 Figure 32 .................................................................................................. 122 Figure 33 .................................................................................................. 123 Figure 34 .................................................................................................. 124. vi.

(9) ABBREVIATIONS 2D. Two-Dimensional. 2-DE. Two-Dimension Electrophoresis. ACN. Acetonitrile. AGC. Auto Gain Control. ATP. Adenosine Triphosphate. BSA. Bovine Serum Albumin. CAB39L. Calcium Binding Protein 39-like. CID. Collision-Induced Dissociation. CRAMP. Cathelin-Related Antimicrobial Peptide. DEPC. Diethylpyrocarbonate. DIGE. Difference Gel Electrophoresis. dNTP. Deoxynucleoside Triphosphates. DTT. Dithiothreitol. ERLIC. Electrostatic Repulsion Hydrophilic Interaction Chromatography. ESI. Electrospray Ionization. FA. Formic Acid. FDR. False Discovery Rate. GRAVY. Grand Average of Hydropathy. HILIC. Hydrophilic Interaction Liquid Chromatography. HPLC. High Performance Liquid Chromatography. IAA. Iodoacetamide. ICAT. Isotope-Coded Affinity Tag. vii.

(10) IEF. Isoelectric Focusing. IL-1β. Interleukin-1 Beta. IL-6. Interleukin 6. IPG. Immobilized pH Gradient. Irg-1. Immunoresponsive Gene 1. ITGA6. Integrin, Alpha 6. iTRAQ®. Isobaric Tags for Relative and Absolute Quantification. kDa. kilodaltons. LC. Liquid Chromatography. LC-MS/MS. Liquid Chromatography-Tandem Mass Spectrometry. MD. Multi-Dimensional. mRNA. Messenger RNA. MS. Mass Spectrometry. MSU. Monosodium Urate. NAD+. Nicotinamide Adenine Dinucleotide. NALP3. NACHT, LRR and PYD Domains-Containing Protein 3. NCE. Normalized Collisional Energy. NF-κB. Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells. NLR. NOD-Like Receptor. PAGE. Polyacrylamide Gel Electrophoresis. PBS. Phosphate-Buffered Saline. pI. Isoelectric Point. PMSF. Phenylmethylsulfonyl Fluoride. PQD. Pulsed-Q Dissociation. viii.

(11) PUMA-g. Protein Upregulated in Macrophages by Interferon-Gamma. PVDF. Polyvinylidene Fluoride. RPLC. Reversed-Phase Liquid Chromatography. RT-qPCR. Quantitative Real Time Polymerase Chain Reaction. SCX. Strong Cation Exchange. sIEF. Solution Isoelectric Focusing. SILAC. Stable Isotope Labeling by Amino Acids in Cell Culture. TCA. Tricarboxylic Acid. TCTP. Translationally-Controlled Tumor Protein. TMT™. Tandem Mass Tag. TNF-α. Tumor Necrosis Factor-Alpha. TPT1. Tumor Protein, Translationally-Controlled 1. TREM-1. Triggering Receptor Expressed on Myeloid Cells 1. Tris. Tris-(hydroxymethyl)-Aminomethane. UV-Vis. Ultraviolet-Visible Spectrophotometry. ix.

(12) ABSTRACT Proteomics is a large-scale comprehensive study of a specific proteome, including information on the levels of different types of proteins, their modifications and variations, as well as their interactions and networks, in order to understand biological processes. Recent successes clearly show that mass spectrometry-based proteomics as an essential tool for molecular and cellular biology and for the rising field of systems biology. Two-dimensional fractionation is a useful tool to increase proteome coverage and the dynamic range than single-dimensional LC. In part I of this dissertation, various peptide fractionation strategies that are used for 2D (two-dimensional) separations were evaluated. The use of SCX x RPLC for desalted samples provided superior results in protein identification. These approaches are complementary and allowed 43% more peptides to be identified, when compared with a single fractionation strategy. In part II, LTQ-PQD parameters were optimized in order to used isobaric tags technology for quantitative proteomics. The number of microscans and the target value are the most critical factors in producing intense reporter ions for quantitation. The appropriate normalized collisional energy range for PQD could be very narrow and must be carefully determined. The optimized LTQ-PQD parameters were introduced to a murine air pouch membrane in part III. iTRAQ-based approach coupled with offline 2D LC-MS/MS proteomics technology was applied to analyze the protein expression profile using an inflamed murine air pouch membrane as a model. Statistical analyses revealed that 317 proteins are differentially expressed, at least at one time point, after the MSU treatment, that they are mainly. x.

(13) involved in the complement system and activation of NALP3 inflammasome. Moreover, the TCA cycle was found to be down-regulated at both the translational and transcriptional levels. Lastly, pyruvate carboxylation was found to be a potential target for an anti-gout treatment. These results provide novel insights into the nature of gouty inflammation.. Keywords: Two-dimensional separation, LC-MS/MS, Pulsed-Q dissociation, Differential proteomics, Monosodium urate crystal, Gout. xi.

(14) INTRODUCTION Proteomic analyses of biological systems represent extreme challenge, due to the great complexity and wide dynamic range of the samples, which can contain hundreds to thousands of proteins or peptides.1-3 The ideal proteome analysis must be capable of identifying and quantifying thousands of proteins over several orders of magnitude. As a result, highly efficient separation techniques and sophisticated instrumentation for detection are required. The combination of a highly efficient separation technique and mass spectrometry (MS) is frequently used in areas ranging from biological function to drug development.4-6. Peptide Fractionation Despite the recent progress in column and instrumentation technologies, liquid chromatography (LC) still lacks the resolving power required for separating diverse and complex biological samples in a single run.3 Since the separation of intact proteins by LC and their identification by MS is difficult, an LC-MS analysis is typically performed on a peptide level, after converting the protein into a series of peptide fragments by treatment with a proteolytic enzyme. This process further amplifies the complexity of the sample, which increases the need for more effective separation methods. All shotgun analyses of an entire proteome still suffer from significant undersampling, since too many co-eluting peptides simultaneously enter the ion source; ionization is suppressed and peptides are sampled at random, all of which result in the under-representation of needed information. Peptide prefractionation plays a critical role in comprehensive proteome analysis. 1.

(15) strategies. Coupled with MS peptide sequencing strategies, prefractionation can substantially increase the number of identifiable components in a proteome.. Two-dimensional. liquid. chromatography. (2D. LC). and. multi-dimensional (MD LC) are frequently utilized to reduce sample complexity and improve separation prior to tandem mass spectrometric analysis. Reversed-phase liquid chromatography (RPLC) is the method of choice for separating complex peptide mixtures in the last dimension of LC separation for a mass spectrometric analysis. This technique is compatible with electrospray ionization. interfaced. with. tandem. mass. spectrometry. for. protein. sequencing/identification. Since reversed-phase chromatography is typically used as the second-dimensional separation prior to mass spectrometric analysis, improvements in 2D LC primarily depend on the first dimension of separation. The success of the multidimensional separation scheme is determined by two major factors: the orthogonality of the separation dimensions and the chromatographic efficiency of the separation systems employed in the different dimensions.7,8 The term orthogonality in chromatography often refers to alternative selectivity between different separation schemes.9 A number of separation modes are available for use in conjunction with RPLC tandem MS analysis, including ion exchange (IEX),10,. 11. hydrophilic. interactions (HILIC),12 isoelectric focusing (IEF),13 and mixed-mode pH reverse phase (RP-RP).14 Among the various forms of IEX, a popular choice relies on the use of a strong cation exchange (SCX) material, which permits peptides to be separated based on the charges of the peptides; they are eluted using a salt gradient or by changes in the pH of the eluent.15 Current 2D LC approaches for. 2.

(16) proteomic analysis frequently utilize SCX chromatography and followed by RP-HPLC coupled to tandem mass spectrometric analysis that delivers sufficient separation of peptides orthogonally.11 Nevertheless, recent reports suggest that the orthogonality of the SCX and RP modes might not be ideal, since the separation efficiency for ion-exchange LC is somewhat limited and peptide clusters of the same charge (mostly +2 and +3) are eluted in a relatively narrow retention window.11, 16 A potential alternative to SCX-based separation is the use of IEF as a first-dimensional separation technique. The OFFGEL. Fractionator. from. Agilent. Technologies,. a. preparative-scale. solution-isoelectric focusing (sIEF) instrument, is commercially available and can be adapted for use at the peptide level.17 The separation is based on conventional IEF but when IPG strips are used in a liquid phase, proteins or peptides can be fractionated according to their pI values, which represent an efficient and reproducible separation approach.18 The four-part diagram in Figure 1 shows starting and ending states, as well as the OFFGEL fractionation process itself. sIEF has several strengths for peptide analysis, including superior resolution, a wide range of loading capacity, high sample recovery and good reproducibility with reasonable separation times.13,17,19 RPLC using high-pH fractionation has been combined with RPLC tandem MS performed with acidic eluents.20,. 21. The orthogonality of high- and low- pH RPLC separations is. adequate, although not perfect, due to the high column separation efficiency provided by both the alkaline and acidic conditions of RPLC.14,22 Since peptides are generally amphoteric, a significant difference in chromatographic selectivity was observed due to changes in charge at the alkaline and acidic pH conditions used for peptide separations.23 HILIC, a technique that has been in. 3.

(17) use for decades, is an analog of normal phase LC (NPLC) and involves the use of a hydrophilic stationary phase and an aqueous mobile phase with a high percentage of organic solvent.24 In HILIC chromatography, the order of elution is reversed relative to RP, with hydrophilic peptides being retained longer than hydrophobic ones. It has been reported that mixed-mode effects consisting of electrostatic and polar interactions also contribute to retention in HILIC.25 Zwitterionic. HILIC. (ZIC-HILIC). describes. mixed. HILIC-ion-exchange. chromatography for the separation of peptides using a cation-exchange sorbent as the first dimension for the analysis of complex peptide mixtures. 26 A variant of HILIC, termed electrostatic repulsion hydrophilic interaction chromatography (ERLIC), was first introduced for the isolation of phosphopeptides,27 but was further applied for use as the first dimension of peptide fractionation in shotgun proteomics.28 A schematic representation of these chromatographic methods is illustrated in Figure 2. The goal of first part of the study was to evaluate the selectivity of several LC fractionation procedures in the first dimension of 2D LC tandem MS analysis and to propose suitable strategies for the fractionation of peptides. LC schemes were critically examined that are compatible with conventionally employed acidic RP LC-MS detection methods, including strong cation exchange (SCX), solution-isoelectric focusing (sIEF),. alkaline-RP, and. hydrophilic interaction chromatography (HILIC). The separation orthogonality, the amounts of peptide/protein identification in each scheme, and complementarities between multiple 2D LC modes were compared and evaluated.. 4.

(18) Differential Proteomics To provide deep insights into the biological processes of life, not only the composition but also the quantities of proteins in biological systems need to be studied. Differential proteomics, the comparison of distinct proteomes such as different physiological or pathological states, is of great importance. Several techniques are available for achieving this, including gel-based approaches and shotgun proteomic methods. The high-resolution 2-DE based separation of protein mixtures followed by image analysis for relative protein quantitation and mass spectrometric analysis for identification is a popular methodology in the field of proteomics.29 Aside of its popularity, 2-DE suffers from low sensitivity, low dynamic range, difficulty in resolving proteins with extreme masses or isoelectric points, and the inability to resolve proteins with a low solubility (membrane proteins, hydrophobic proteins). Labeling a protein with a fluorescent dye, as introduced during the DIGE technology, partly overcomes the limitations associated with 2-DE.30 Stable isotope labeling and label-free approaches are the two strategies used for quantification in shotgun proteomic methods. Label-free approaches fall into two categories: peak area of peptides and spectral counting. Although label-free method can be used conveniently for large scale proteome quantification, quantification accuracy is suspicious due to variations introduced during sample preparation and LC-MS/MS analysis. This challenge can be relieved by stable isotope labeling based quantification methods. Stable isotope labeling can be introduced metabolically (SILAC),31 enzymatically (O16/O18)32 or chemically (ICAT),33 iTRAQ®34 and TMT (Tandem. 5.

(19) Mass Tag™).35 Among the chemical labeling approaches, isobaric tags for relative and absolute quantitation (iTRAQ) has received more attention due to its multiplexing, higher numbers of identified peptides and lower variations of the quantitative results.36, 37. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) The iTRAQ technology uses isobaric reagents to label peptides thus enabling quantitation up to four or up to eight different biological samples within a single experiment. The reagents consist of a N-methyl piperazine reporter group, a balance group, and a N-hydroxy succinimide ester group that reacted with the primary amines of peptides. Due to its isobaric mass design of the iTRAQ reagents, labeled peptides are indistinguishable in the MS survey scan. In the MS/MS scan, the peptide-linked tag can fragment resulting in the neutral loss of the balance group, release of a reporter ion for protein quantitation, and b- and y- types of ions for protein identification. Furthermore, this technology can be multiplexed for the parallel analysis of four samples, and the method also permits multiple independent measurements to be made for the relative abundance of any given protein. Among the challenges associated with this and other quantitative shotgun approaches, it is possible that a particular proteolytic peptide may be derived from different proteins and the MS/MS spectra from multiple precursors from a given protein may result in significantly different quantifications for that particular protein.38 The multiplexed derivatization chemistry and the experimental workflow are introduced in Figure 3.. Pulsed-Q Dissociation (PQD) In an iTRAQ experiment, quantification is based on the relative intensities. 6.

(20) of reporter ions which appear in the low mass range of MS/MS spectra. Mass spectrometers equipped with tandem in space design such as quadrupole time-of-flight instruments have the inherent capability of detecting low m/z fragment ions in contrast to ion traps (tandem in time). Ion traps have one limitation, commonly known as the “one-third rule”,39 arising from the recovery of fragments ion that are less than 30% of the precursor ion which selected for fragmentation by CID during MS/MS analysis. A previously introduced the use of an activated fragmentation technique subject to pulsed-Q dissociation (PQD) eliminates the one-third rule for ion trap mass spectrometers and makes the analysis of iTRAQ-labeled samples possible with these instruments, as PQD enables routine and reliable measurement of ions down to 50 m/z.40 PQD involves 3 discrete stages of operation of the linear ion trap: (1) activation of the precursor at high Q to allow precursors to rapidly gain kinetic energy (7 times higher than that in CID mode), (2) after excitation, the precursor ions gain enough kinetic energy, begin colliding with helium, thus convert kinetic energy into internal energy and lastly (3) a pulse to low Q to trap all of the fragment ions generated after the collisions.41 In this part of the study, instrument parameters including number of trap ions (target value), number of microscans, normalized collisional energy, activation Q, and delay time were carefully optimized. As a result, low m/z fragment ion intensities can be generated that enable accurate peptide quantification at the femto-mole level.42, 43. Monosodium Urate Crystals-Induced Responses in Dissected Murine Air Pouch Membranes Crystal deposition has long been a topic in arthritis research.. 7.

(21) Monosodium urate (MSU) crystals (NaC5H3N4O3 ·H2O) are a stimulating agent for the development of gout, which is one of the most common causes of inflammatory arthritis in men. Monosodium urate crystals are a white powdery substance and display needle-like crystalline symmetry under the microscope. The appearance of tophi has been described by many historians and artists throughout history. However, it was not until 1849 when Alfred B. Garrod, a British physician, linked high levels of uric acid with gouty arthritis. 44 Garrod’s hypothesis was later confirmed in 1962 when Faires and MacCarty injected MSU crystals into healthy human and canine joints and reproduced the typical symptoms of gout.45 Multiple mechanisms have been proposed for the spontaneous resolution of acute gout, including the coating of crystals with protein receptors and changes in the balance of expression of proinflammatory and anti-inflammatory factors as the cell population within the inflamed joint.46 It is believed that MSU crystals stimulate macrophages and monocytes to secret inflammatory cytokines, but the protein profile of the synovium is not fully understood. The murine air pouch is an easily obtainable model, which is produced de novo in the dorsal subcutaneous tissue.47 Within 2 to 5 days by subcutaneous injection of 3 mL of filtered air, a membrane comprised of several layers of cells grows around this air-filled space. This membrane consists mostly of fibroblasts, mononuclear cells and small blood vessels and, histologically, resembles the synovial membrane. The synovial lining possesses some important properties, such as the expression of the Ia antigen and the formation of hyaluronic acid. Micro-organisms or inflammatory stimulating agents can be easily injected into the air pouch, trigger different types of. 8.

(22) immune response. Inflammation induced by MSU crystals has been studied utilized this model.48, 49 Protein profiling of intact tissues could be obstructed by the adjacent non-inflamed area, which increases the complexity of the tissue and can lead to negative effects on the responses. To minimize the sample complexity, the dissected membrane from the overlying subcutaneous tissues was used to study whole-tissue protein profile in an inflamed tissue.48 In a previous study, Pessler et al.48 reported on mRNA microarray analyses of the equivalent of synovial lining cells after MSU crystals had been injected into a murine air pouch; their study addressed the tissue response to gouty inflammation at 9 hours after injection of MSU crystals. mRNA microarrays technology is widely applied and allows simultaneous measurement of the mRNA levels of thousands of genes. However, many groups have used data regarding changes in gene expression as evidence of consequent changes in protein expression. Despite the fact that these data are useful, mRNA levels cannot be used as surrogates for corresponding protein levels without verification. Previous study shows that the most serious and interesting regulators of cellular function and differentiation would be expected to have a poor correlation between mRNA and protein while housekeeping proteins would probably have relatively good correlation.50 In essence, proteins are effector molecules and regulate biological functions in the body, therefore, measuring these, in contrast with mRNA expression, can lead to a better understanding of disease processes as well as drug treatment and therapeutics. To address this research gap, iTRAQ labeling coupled with offline 2D LC-MS/MS proteomics technology was applied to analyze the protein. 9.

(23) expression profile using an inflamed murine air pouch membrane as a model. The objective of this study was to first establish a proteomics platform for identifying proteins in the inflamed murine air pouch membrane, and second to quantify specific candidate proteins that could serve as a novel metabolic target for the treatment of gout.. 10.

(24) EXPERIMENTAL SECTION Chemicals All chemicals were analytical grade or better, and were purchased from Sigma Aldrich (St. Louis, MO, USA). Chromatography grade acetonitrile was obtained from Merck KGaA (Darmstadt, Germany). Bradford protein assay materials were purchased from Bio-Rad (Hercules, CA, USA). Porcine trypsin modified (sequencing grade) was obtained from Promega (Madison, WI, USA). The iTRAQ 4-plex reagent kits were purchased from AB SCIEX (Foster City, CA, USA). Desalting C18 spin columns were purchased from Pierce (Thermo Fisher Scientific, Rockford, IL, USA). Centrifugal filters were purchased from Millipore (Merck KGaA, Darmstadt, Germany). Column packing materials for the analytic column were purchased from Macherey Nagel (Düren, Germany). Packing materials for the trap column were purchased from Michrom Bioresources (Auburn, CA, USA). Protein standard of bovine serum albumin (BSA), lysozyme, myoglobin, cytochrome C, and phosphorylase B were purchased from Sigma Aldrich. Sequencing grade modified trypsin was purchased from Promega (Madison, WI, USA).. Sample Preparation and Digestion of PLC/PRF/5 lysate The human hepatoma cell line PLC/PRF/5 lysates were generously provided by Dr. Ko, of the Industrial Technology Research Institute, Taiwan. The cell lysate was passed through a 3 kDa weight cutoff filter (Amicon ® Ultra-0.5 Millipore, Bedford, MA, USA). The amount of protein in the retentate was determined by the Bradford protein assay, and the samples were stored at -20 °C for subsequent processing.. 11.

(25) The retentate was diluted to 1 µg of lysate/1 µL in 50 mM NH4HCO3. The sample was then reduced by treatment with 23.5 µL of 200 mM dithiothreitol (DTT) in 100 mM NH4HCO3 and heated at 95 oC for 5 min. The solution was cooled to room temperature for 10 min and alkylated by treatment with 18.8 µL of 1 M iodoacetamide (IAA) in 100 mM NH4HCO3 for 45 min in the dark at room temperature. The alkylation reaction was stopped by adding 94 µL of 200 mM DTT in 100 mM NH4HCO3, followed by incubation for 45 min. Trypsin was added at a ratio 1:50 (trypsin : sample) at 37 oC for a 16 h period of digestion. The peptides were concentrated in a SpeedVac and then dissolved in 0.1% formic acid (FA) in 2% ACN for desalting using a reverse-phase HPLC.. Protein Standard Mixture Preparation and iTRAQ Reagent Labeling for LTQ-PQD optimization iTRAQ-labeled protein mixtures of bovine serum albumin (BSA), lysozyme, myoglobin, cytochrome C, and phosphorylase B was used to optimize the instrument parameters for LTQ-PQD. Protein denaturation, digestion, and iTRAQ. labeling. were. performed. according. to. the. manufacturer’s. recommendations (AB SCIEX, Foster City, CA, USA). Briefly, protein mixtures were dissolved in 20 μL of dissolution buffer (20 mM triethylammonium bicarbonate, TEAB), followed by the addition of 1 μL of 2% sodium dodecyl sulfate (SDS). This was followed by the addition of 2 μL of reducing agent (50 mM Tris (2-carboxyethyl) phosphine, TCEP). The samples were incubated at 60 °C for 1 h, 1 μL of alkylating agent (200 mM methyl methane-thiosulfonate, MMTS) was added as a cysteine-blocking agent, and the samples were incubated for an additional 10 min at room temperature. Samples were then incubated with trypsin (20/1, w/w) overnight at 37 °C. After reduction,. 12.

(26) alkylation and tryptic digestion, the peptides were divided into four different tubes; each tube contained 10, 50, 50 and 100 μg of total peptides. Samples were iTRAQ labeled as follows: 50 μg, 114; 10 μg, 115; 50 μg, 116; 100 μg, 117. The labeled peptide samples were then pooled and lyophilized in a vacuum concentrator and reconstituted for LC-MS/MS analysis. The iTRAQ ratio of 114:115:116:117 was 5:1:5:10.. Murine Air Pouches Air pouches were prepared as described by Jung et al.48 Briefly, air pouches were introduced on the backs of 6- to 8-week-old male BALB/c mice (BioLASCO Taiwan Co., Ltd.) by the subcutaneous injection of 2 mL of filtered air on day 0. A second injection was given on day 3 with an additional 3 mL of filtered air to keep the air pouch inflated. MSU crystals (U-2875, Sigma) were ground and sieved through a 120-mesh screen, and then aliquoted from the same batch and heated at 180 °C for 2 h to eliminate endotoxins. A suspension of 3 mg MSU crystals in 2 mL of sterile endotoxin-free saline was injected into 6-day-old air pouch to induce inflammation. The negative control group was injected with the same volume of saline and harvested at 1 h after injection. To verify the time points of the peak and natural resolution of inflammation in the pouch, a 5 h time course experiment was performed (Figure 4A). Animals were sacrificed at time points after the injection of MSU crystals and during which 2 mL of a sterile endotoxin-free saline was injected into the pouch. The pouch exudate was collected for cytokine analysis and leukocyte counting and the pouch membrane was collected for protein extraction. In the cytokine array experiment, the control pouch exudate (n=4) and inflamed pouch exudate (n=5) at each time point were separately centrifuge at 1000 rpm, at 4. 13.

(27) °C for 5 min and the supernatants were harvested for IL-1β (interleukin-1 beta), IL-6 (interleukin 6), and the determination of TNF-α (tumor necrosis factor-alpha) levels using commercial ELISA kits (DuoSet® R&D, Minneapolis, MN, USA). Leukocyte counting experiments used the same sample as those used in the cytokine array. All of the experimental protocols involving experimental animals and their care were approved by the Institutional Animal Care and Use Committee of Industrial Technology Research Institute and were carried out according to the regulations of the Council of Agriculture, Taiwan.. Dissection of the Air Pouch Membrane After sacrificing the animals by carbon dioxide euthanasia, the pouch membranes were carefully dissected using a previously described method (Figure 4B).48 Briefly, a T-shaped incision was made into the dorsal skin overlying the pouch by scissors to expose the apex of the pouch membrane. The pouch membrane was then carefully separated from the overlying skin and adjacent subcutaneous and paraspinal tissues by blunt dissection. Finally, the membrane was grasped with forceps, elevated and cut at the base with scissors. The isolated membranes were frozen immediately in liquid nitrogen and stored at -80 °C until further use.. Air Pouch Membrane Protein Extraction Frozen MSU treated murine synovial membranes were ground thoroughly in a mortar cooled with liquid nitrogen. Negative control pouch membranes (n=2) and inflamed pouch membranes (n=3) at each time point were pooled respectively for protein extraction in iTRAQ experiment. The tissue powder was homogenized, resuspended in RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing 1X. 14.

(28) protease inhibitor cocktail (Roche, Switzerland), 1 mM PMSF and kept on ice for 20 min. After centrifugation at 14 000 × g, at 4 °C for 10 min, the supernatants were collected and stored at -80 °C until use.. Murine Air Pouch Sample Pretreatment Preparation and iTRAQ Reagent Labeling For each time point, proteins were concentrated using a 3 kDa centrifugal filter, as described by the manufacturer. Briefly, 100 L samples were centrifuged at 14 000 × g for 20 min at 4 °C in the filter tube. The filter device was placed upside down in a clean tube and allowed to spin for 2 min at 1000 x g. This process was repeated twice using ultrapure water for desalting and to remove the protease inhibitor cocktail. The amount of protein in each concentrated/desalted sample was determined by the Bradford protein assay, and were then stored at -20 °C for subsequent processing. An overview of the experimental strategy is shown in Figure 5. Briefly, the extracted proteins were desalted and quantified by means of a Bradford protein assay. Equal amounts of protein were first digested and labeled with different iTRAQ tags. The labeled digests were then mixed and prefractionated via SCX, alkaline-RP and solution-IEF before the LC-MS/MS analysis.51 An Agilent 1200 instrument and a LTQ-XL system were used as data-dependent acquisition platforms. Twenty microliters of iTRAQ dissolution buffer (500 mM triethyl ammonium bicarbonate) was added to a dried sample containing 100 μg of each sample for protein solubilization. Proteins were then reduced and alkylated using iTRAQ kit reagents, following the manufacturer’s instructions. Samples were digested for 16 h at 37 °C with 3 μg trypsin. The samples were labeled as follows: control, iTRAQ 114; MSU injection for 1 h, iTRAQ 115; MSU. 15.

(29) injection for 3 h, iTRAQ 116; MSU injection for 5 h, iTRAQ 117. After 1 h of iTRAQ labeling, the samples were then mixed and dried by centrifugal evaporation.. Analytical Setups for Peptide from the PLC/PRF/5 Lysate Pretreatment The RP-HPLC setup consisted of an Agilent 1100 series HPLC instrument equipped with a manual injector (Model Rheodyne 7725, Oak Harbor, WA, USA). Approximately 300 µg of peptides from the PLC/PRF/5 lysate were injected into a Zorbax XDB-C18 column (150 x 4.6 mm, 5 µm, 80 Å; Agilent, Waldbronn, Germany). All runs were operated at 0.6 mL/min, and the column was maintained at 30 oC. The RP-HPLC mobile phase consisted of mobile phase A (0.1% formic acid in 2% ACN) and mobile phase B (0.1% formic acid in 98% ACN). The gradient was 5% mobile phase B for 30 min, 5-80% mobile phase B for 1 min, followed by 30 min at 80% mobile phase B, and the fraction collected within the last 30 min was retained. After collecting the desalted peptides, they were pooled and a 155 µg aliquot used for the downstream analysis. (Figure 6). Analytical Setups for First-Dimension Separation for the PLC/PRF/5 Lysate Sample All LC fractionation experiments were performed using an Agilent 1100 Series instrument. The HPLC system consisted of a binary pump, a vacuum degasser, an autosampler, a UV/Vis detector set as 214 nm, and a manual injector with a home-calibrated loop (200 µL or 2 mL). The column temperature was maintained at 30 oC by the column oven to assure the reproducibility in the experiments. The 24 fractions acquired from all five separation strategies were further desalted by C18 spin column before. 16.

(30) LC-MS/MS analysis. The two-dimensional separation scheme is summarized in Table 1.. SCX Chromatography Tryptic peptides from 155 µg of protein (PLC/PRF/5 cells) were fractionated on a PolySULFOETHYL A column (2.1 x 200 mm, 5 µm, 300 Å, PolyLC INC.) with a Javelin guard cartridge guard column (2.1 x 10 mm, 5 µm, 300 Å, PolyLC INC.). Desalted sample was reconstituted in 180 µL of mobile phase A and non-desalted sample was reconstituted in 1.8 mL of mobile phase A then loaded into the column. The mobile phases used were composed of (A) 10 mM KH2PO4 in 25% ACN, pH 2.9 and (B) 10 mM KH2PO4 with 350 mM KCl in 25% ACN, pH 2.9. The mobile phases were applied using a gradient of 2% B for 5 min, 2–50% B for 40 min, 50–98% B for 5 min, 98% B for 5 min followed by 98-2% B for 5 min and then maintained at 2% B for 10 min at a flow rate of 0.2 mL/min. The elution was monitored by absorbance at 214 nm, and fractions were collected every 1 min. Finally, these samples were combined into 24 fractions based on the quantity of peptide and then desalted on C18 spin column. (Figure 7-11) One hundred twenty micrograms of the mixture of iTRAQ-labeled peptides from murine air pouch sample was reconstituted with 1.8 mL of mobile phase A. The separation was performed using a PolySULFOETHYL A Column (200 mm L × 2.1 mm i.d., 5 µm, 300 Å, PolyLC, Columbia, MD) on an Agilent 1100 binary HPLC system (Agilent Technologies, Wilmington, DE, USA). The mobile phases used were composed of (A) 10 mM KH2PO4 in 25% ACN, pH 3.0 and (B) 10 mM KH2PO4 with 350 mM KCl in 25% ACN, pH 3.0. The mobile phases were applied using a gradient of 2% B for 15 min, 2–40% B for 38 min,. 17.

(31) 40–98% B for 7 min, 98% B for 5 min followed by 98-2% B for 5 min and then maintained at 2% B for 20 min at a flow rate of 0.2 mL/min. The elution was monitored by absorbance at 214 nm, and fractions were collected every 2 min. Finally, these samples were combined into 24 fractions based on the quantity of peptide and then desalted on C18 spin column (Thermo Fisher, San Jose, CA, USA) for further nano LC-MS/MS analysis.. HILIC Chromatography Tryptic peptides from 155 µg of protein (PLC/PRF/5 cells) were fractionated on an Atlantis® HILIC silica column (2.1 x 100 mm, 3 μm, 100 Å, Waters). The mobile phases used were composed of (A) 8 mM NH4HCO2 in 30% ACN, pH 4.5 and (B) 8 mM NH4HCO2 in 80% ACN, pH 4.5. The mobile phases were applied using a gradient of 98% B for 5 min, 98–50% B for 45 min, 50–10% B for 5 min, 10% B for 10 min followed by 10-98% B for 5 min and then maintained at 98% B for 10 min at a flow rate of 0.2 mL/min. The elution was monitored by absorbance at 214 nm, and fractions were collected every 1 min. Finally, these samples were combined into 24 fractions based on the quantity of peptide and then desalted on C18 spin column.. Reverse Phase Chromatography at Low pH Tryptic peptides from 155 µg of protein (PLC/PRF/5 cells) were fractionated on a Biobasic C18 column (2.1 X 150 mm, 5 μm, 300 Å, Thermo Scientific INC.). The mobile phases used were composed of (A) 0.1% FA in 2% ACN and (B) 0.1% FA in 98% ACN. The mobile phases were applied using a gradient of 2% B for 3 min, 2–60% B for 50 min, 60–98% B for 2 min, 98% B for 10 min followed by 98-2% B for 3 min and then maintained at 2% B for 12 min at a flow rate of 0.2 mL/min. The elution was monitored by absorbance at 214. 18.

(32) nm, and fractions were collected every 1 min. Finally, these samples were combined into 24 fractions based on the quantity of peptide and then desalted on C18 spin column.. Reverse Phase Chromatography at High pH Tryptic peptides from 155 µg of protein (PLC/PRF/5 cells) were fractionated on a Biobasic C18 column (2.1 X 150 mm, 5 μm, 300 Å, Thermo Scientific INC.). The mobile phases used were composed of (A) 50 mM Tris in 2% ACN, pH 8.5 and (B) 50 mM Tris in 80% ACN, pH 8.5. The mobile phases were applied using a gradient of 2% B for 3 min, 2–74% B for 50 min, 74–98% B for 2 min, 98% B for 10 min followed by 98-2% B for 3 min and then maintained at 2% B for 12 min at a flow rate of 0.2 mL/min. The elution was monitored by absorbance at 214 nm, and fractions were collected every 1 min. Finally, these samples were combined into 24 fractions based on the quantity of peptide and then desalted on C18 spin column. One hundred twenty micrograms of the mixture of iTRAQ-labeled peptides from murine air pouch membrane was reconstituted with 180 μL of mobile phase C. The separation was performed using a Biobasic-18 column (150 mm L x 2.1 mm i.d., 5 μm, 300 Å, Thermo Fisher, San Jose, CA, USA) on an Agilent 1100 binary HPLC system. The mobile phases used were composed of (C) 50 mM NH4OH in 2% ACN at pH 8.4 and (D) 50 mM NH4OH in 80% ACN at pH 8.4. The mobile phases were applied using a gradient of 2% D for 3 min, 2– 60% D for 50 min, 60–100% D for 2 min, 100% D for 10 min followed by 100-2% D for 3 min and then maintained at 2% D for 12 min at a flow rate of 0.2 mL/min. The elution was monitored by absorbance at 214 nm, and fractions were collected every 2 min. Finally, these samples were combined. 19.

(33) into 24 fractions based on the quantity of peptide and then desalted on C18 spin column for further analysis.. Solution-IEF Separation Tryptic peptides from 155 µg of protein (PLC/PRF/5 cells) were fractionated using a 3100 OFFGEL Fractionator (Agilent Technologies, Waldbronn, Germany) according to the manufacturer’s protocol. An Agilent 3100 OFFGEL fractionator kit and 24-lane gel strips with a linear pH in the pH range of 3-10 were used. The 24-lane gel strip was rehydrated with 40 µL of focusing buffer per well. All samples were dissolved in 3.6 mL of focusing buffer. One hundred and fifty microliters of sample buffer were added to each well. A standard tray method (OG24PE48) was applied for the fractionation. After focusing, the 24 peptide fractions were withdrawn and the wells were rinsed with 200 μL of an ultrapure water/methanol/formic acid (49/50/1) solution. After 15 minutes, each of the rinsing solutions was pooled with the corresponding peptide fractions. All of the fractions were desalted using a C18 spin column, and concentrated using a SpeedVac. One hundred twenty micrograms of the mixture of iTRAQ-labeled peptides from murine air pouch membrane was dissolved in 0.72 mL ultrapure water and 2.88 mL of IPG stock buffer (pH 3-10). The IPG strip (pH 3-10, 24 cm) was assembled on the OGE trays and rehydrated for 15 min with a solution of 240 µL ultrapure water and 0.96 mL of IPG stock buffer. The samples were loaded on 24 OFFGEL wells. The separation was carried out 48 h using the 3100 OFFGEL fractionator (Agilent Technologies, Wilmington, DE, USA) with a limiting current of 50 µA and a limit of 50 kV· h before holding the voltage at 500 V. The samples from 24 fractions were cleaned-up using C18 Spin. 20.

(34) columns for further analysis.52. LC-MS/MS Analysis The HPLC setup consisted of an Agilent 1200 series HPLC instrument was coupled online to LTQ XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) with a modified nano-ESI source. The peptides were trapped in a lab-made pre-column consisting of a capillary column (100 µm x 2 cm) packed with C18 AQ (5 µm, 200 Å, Michrom bioresources, Auburn, CA, USA) and separated by an analytical column consisting of a capillary column (75 µm x 10 cm) packed with C18 (3 µm, 100 Å, Macherey-Nagel, Düren, Germany). Each PLC/PRF/5 lysate sample was reconstituted in 40 µL of mobile phase A (0.1% FA in 1% ACN); 20 µL of each reconstituted fraction was and separated with a linear gradient of mobile phase B (99% ACN with 0.1% (v/v) FA) at a flow rate of 250 nL/min over 140 min: 0–5 min with 2% B; 5–100 min with 2-40% B; 100-105 min with 40-80% B; 105-110 min with 80% B; 110-115 min with 80-2% B; 115-140 min with 2% B. A 2.0 kV spray voltage was applied through a liquid junction. The temperature of the ion transfer capillary was set at 200 °C, and the normalized collision energy was at 35.0% with activation times of 30 ms for the LTQ XL. Ions were scanned over the range m/z 350-2000. For data-dependent acquisition, the mass spectrometer was set as one full MS scan followed by zoom scan and MS/MS scan of the three most intense ions in full MS. The dynamic exclusion function was set as follows; repeat count 2, repeat duration 0.5 min, and exclusion duration 3 min. System control and data collection were done by Xcalibur software version 2.1 (Thermo Fisher Scientific, San Jose, CA, USA).. 21.

(35) LC-MS/MS Analysis by LTQ-PQD Experiments were performed on the LTQ-XL (Thermo Fisher, San Jose, CA, USA) that was coupled with an Agilent 1200 series HPLC. The peptides were trapped in a lab-made pre-column consisting of a capillary column (100 µm x 2 cm) packed with C18 AQ (5 µm, 200 Å, Michrom bioresources, Auburn, CA, USA) and separated by an analytical column consisting of a capillary column (75 µm x 10 cm) packed with C18 (3 µm, 100 Å, Macherey-Nagel, Düren, Germany). For LTQ-PQD optimization and murine air pouch membrane experiments, 2 µg and 0.5 µg (est.) peptides was loaded, respectively. And separated at a flow rate of 250 nL/min across the analytical column with a linear gradient of 2-40% mobile phase B (0.1% formic acid in 99% ACN) for 95 min, 40-80% B for 5 min and then maintained in 80% B for 5 min before equilibrating with 2% B for 25 min. A 2.0 kV spray voltage was applied through a liquid junction. The temperature of the ion transfer capillary was set to 200 °C. Ions were scanned over the range m/z 350-2000. The LTQ-XL was operated in a data-dependent mode, that is, one MS1 scan for precursor ions followed by three data-dependent zoom scans for precursor ions above a threshold ion count of 200 000, and then followed by three data-dependent PQD-MS2 scans and CID-MS2 scans for precursor ions in zoom scan above a threshold ion count of 100 with the normalized collision energy was at 35.0% with activation times of 30 ms for the CID. The dynamic exclusion function was set as follows; repeat count 2, repeat duration 0.5 min, and exclusion duration 3 min. System control and data collection were done by Xcalibur software version 2.1 (Thermo Fisher Scientific, San Jose, CA, USA).. 22.

(36) Peptide and Protein Identification and Quantitation Protein identification of PLC/PRF/5 lysate were performed with MS/MS data using the MASCOT search engine (v.2.3.2, Matrix Science, London, UK) embedded into Proteome Discoverer software (Thermo Fisher, version 1.1.0.263). The search was performed against the Human IPI v.3.84 database (90 166 sequences; 36 304 241 residues; Jun 2011) with search parameters including: enzyme, trypsin; max. miss cleavages, 1; fixed modifications, carbamidomethyl (C); dynamic modification, oxidation (M); MS peptide tolerance as 1.5 Da; MS/MS tolerance as 0.6 Da. For phosphopeptide identification, phosphorylation on serine, threonine, and tyrosine were selected as variable modifications. A decoy database search strategy was utilized to estimate the FDR (false discovery rate) for peptide identification. Proteins with at least two peptides (p <0.05) were used for identification. The resulting MSF files from Proteome Discoverer were sent to Scaffold (version 3.3.1, Proteome Software Inc.) for created the Venn diagrams of proteins and peptides. Unless otherwise specified, all statistics and data normalization were calculated using Microsoft Excel. Protein identification and quantification for the standard protein mixture iTRAQ samples were performed with MS/MS data using the MASCOT search engine (v.2.3.2, Matrix Science, London, UK) via Proteome Discoverer software (Thermo Fisher, version 1.1.0.263). The search was performed against the SwissProt (version 2011_02, 525207 sequences) with search parameters including: taxonomy, mammalian (for standard protein mixture), Homo sapiens (for oral cancer sample); enzyme, trypsin; max. miss cleavages, 1; fixed modifications, methylthiolation, N-terminal iTRAQ 4-plex, lysine iTRAQ 4-plex;. 23.

(37) variable modifications, methionine oxidation, tyrosine iTRAQ 4-plex; MS peptide tolerance as 1.5 Da; MS/MS tolerance as 0.6 Da. Proteins with at least two peptides (p <0.05) were used for identification. All quantitative results were calculated based on the application of default bias-corrections. Quantitative variables were analyzed by the Proteome Discoverer software. Unless otherwise specified, all statistics and data normalization were calculated using Microsoft Excel. Protein identification and quantification of the murine air pouch iTRAQ samples were performed with MS/MS data using the MASCOT search engine (v.2.3.2, Matrix Science, London, UK) embedded into Proteome Discoverer software (Thermo Fisher, version 1.1.0.263). The search was performed against the SwissProt (version 2011_02, 525207 sequences) with search parameters including: taxonomy, Mus.; enzyme, trypsin; max. miss cleavages, 1; fixed modifications, methylthiolation, N-terminal iTRAQ 4-plex, lysine iTRAQ 4-plex; variable modifications, methionine oxidation, tyrosine iTRAQ 4-plex; MS peptide tolerance as 1.5 Da; MS/MS tolerance as 0.6 Da.53 A decoy database search strategy was utilized to estimate the FDR for peptide identification. Proteins with at least two peptides (p <0.05) were used for identification. All quantitative results were calculated based on the application of default bias-corrections. Quantitative variables were analyzed by the Proteome Discoverer software. Unless otherwise specified, all statistics and data normalization were calculated using Microsoft Excel. Differentially expressed proteins were selected based on the following criteria: (1) at least two quantifiable spectra, (2) a fold change ≥1.5 or ≤0.67 and (3) a coefficient of variation (%) lowers than 30%. The molecular functions and pathway. 24.

(38) analysis of the differentially expressed proteins identified in the study were classified using GeneGO MetaCore analysis.. Western Blotting Equal amounts (30 μg) of protein samples were resolved on SDS-PAGE gels and transferred to a PVDF membrane (PerkinElmer, Waltham, MA, USA). The membranes were blocked for 30 min at room temperature with 5% nonfat milk in PBST (0.1% Tween 20 in phosphate buffered saline). The following antibodies were used for Western blot analysis: cathelin-related antimicrobial peptide (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), S100A9 (1:2000, R&D systems, Minneapolis, MN, USA) and beta-actin (1:1000, Epitomics, Burlingame, CA, USA). The membrane probed with primary antibody, and developed using enhanced chemiluminescence detection (PerkinElmer, Waltham MA, USA).. Treatment of THP-1 Cells with a Suspension of MSU Crystals The human acute monocytic leukemia cell line THP-1 (purchased from ATCC, Manassas, VA, USA) was cultured in RPMI-1640 media (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and 1 mM sodium pyruvate. Cells were incubated at 37 °C under humid conditions and a CO 2 level of 5%. To evaluate the inflammatory level induced by MSU crystals the cells were then treated with 0, 50, 100 or 200 μg/mL MSU crystals for 24 or 48 h. The production of IL-1β, IL-6 and TNF-α was detected by quantitative sandwich enzyme immunoassay technique according to the manufacturers’ standard protocols. (Figure 12) The optimum conditions were treated with a 200 μg/mL MSU crystal suspension or PBS only and harvested at 24 h after the treatment. After centrifuging at 1000 x g for 1 min, the cell pellet was washed. 25.

(39) with PBS and then used for total RNA isolation.. Total. RNA. Isolation,. Reverse. Transcription,. Real. Time. PCR. Quantification of Genes Total RNA was isolated from cells using a GeneJET RNA Purification Kit (Thermo. Scientific). according. the. manufacturer’s. instructions.. RNA. concentrations were determined using a NanoDrop 1000 (Thermo Scientific). The ratio of A260/A280 of isolated RNAs was over 1.9. About 1.2 µg of total RNA was then reversed transcribed into cDNA using the ThermoScripTM RT-PCR System for qRT-PCR (InvitrogenTM). The 20 µL RT reaction mixture contained 2 µL of 10 mM dNTP Mix, 1 µL of primer (random hexamers), 8 µL of total RNA, 1 µL of DEPC-treated water and 8 µL of cDNA synthesis mix. The reaction mix was incubated at 65 °C for 5 min and the temperature was increased to 50 °C for 50 min. The reaction was terminated at 85 °C for 5 min and immediately chilled on ice. For gene expression analysis, the qPCR mix was in a final volume of 10 µL and contained 5 µL of Smart Quant Green Master Mix, 1.5 µL of 150 nM each primer and 2 µL of cDNA template (25 ng). The thermal conditions for qPCR were as follows: denaturation; 95 °C for 10 min, 1 cycle, PCR amplification; 95 °C for 15 s (denaturation), 60 °C for 1 min (annealing), 40 cycles, 95 °C for 15 s; 60 °C for 1 min, 95 °C for 15 s, 1 cycle. The assay included a nontemplate control and no reverse transcriptase control for assessing the efficiency of each set of primers and the residue of genomic DNA. All reactions were run in triplicate to reduce experimental variance. The genes that were examined and their primers used in this work are listed in Table 2.. 26.

(40) RESULTS AND DISCUSSION Part I - Evaluation of Peptide Fractionation Strategies used in Proteome Analysis. Two-dimensional LC separation possesses sufficient resolving power to substantially reduce the spatial and temporal complexity of peptide mixtures. An increase in the number of measurable peptides, and widening the overall dynamic range consequently results in an increase in proteome coverage. Four different peptide fractionation strategies for 2D LC MS/MS were compared in this study. The goal of the study was to evaluate which fractionation strategy permits the highest number of peptides/proteins to be identified. The method of choice in the second LC dimension is acidic-RP, because of its high resolution and compatibility with ESI-MS/MS. A flowchart of the experiment is presented in Figure 13, one milligram of the PLC/5 cell lysate was reduced, alkylated and digested, as described in the experimental section. Equal peptide-containing aliquots (155 µg) were first desalted using an RP column and the resulting solution injected first to the LC dimension including acidic-RP, SCX, HILIC, alkaline-RP chromatography or separated on an OFFGEL fractionator. Figure 6 shows the chromatographic profile of the peptide mixture desalted using a C18 column; fractions starting from 36 to 40 min were collected. The first dimension analysis resulted in 24 fractions, typical of a global proteomic analysis,54 which were further analyzed by LC-MS/MS. The acidic-RP was used as a control, since it has the same separation properties as the second LC dimension. The five methods examined were based on previously described methods55-57 as well as our own. 27.

(41) experiences with these separation techniques. The total proteins/peptides identified from each 2D separation method were used to indicate the overall fractionation efficiency. The distribution of the identified peptides and proteins are illustrated in Figure 14. The Influence of Salt on Separation Efficiency A salt gradient was used to elute the peptides from the SCX column, similar to a previous study.58 The peptide sample contained NH4HCO3 from the digestion step. In order to examine the influence of salt content (~10 mM) on the SCX separation, both non-desalted and desalted peptide samples were examined. As shown in Table 3 and Figure 15, a total of 1990 proteins were identified from the desalted sample but only 1375 proteins were identified from the non-desalted sample. This difference was magnified at the peptide level; 10 055 distinct peptides were found in the desalted sample, versus only 4644 peptides in the case of the non-desalted sample. The comparison between the non-desalted and desalted samples for protein/peptide abundance per fraction is shown in Figure 16. It was possible to identify more proteins/peptides in nearly all the fractions of desalted sample than non-desalted samples. This was especially true for fractions after No. 10 where the peptide contained three or more charges. As shown in Figure 17, the charge distribution for the non-desalted sample is quite different with the desalted sample, in that peptides with a charge of +2 were distributed in the first 8 fractions in the desalted sample but only appear in the first 2 fractions in the case of the non-desalted sample. This indicates that peptides with charges of +2 may not be retained in the SCX column when the salt concentration is greater than the KH2PO4 concentration in the mobile phase A. 28.

(42) (10 mM), which causes the peptides to elute with the void volume, without being retained and separated. On the other hand, the identification result from solution-IEF didn’t massively affected by the desalting step. As shown in Figure 16, of the 24 fractions from the non-desalted sample, 15 contained larger numbers of identifiable peptides, compared to the desalted sample. The total number of distinct peptides identified in non-desalted sample was slightly greater than those in the desalted sample in the case of solution-IEF. This indicates that, provided the salt concentration is maintained below 10 mM, as suggested by the manufacturer, the salt content of the sample will not be a critical factor in separation efficiency in the case of solution-IEF. Orthogonality of Two-Dimensional Separations Orthogonality in chromatography refers to alternative selectivity between separations. Orthogonal, or 2D separations are needed to address one of the major concerns in method development, i.e., insufficient resolution, which can mask signals from analytes with similar physical and chemical properties. Such separations can be achieved by modifying some of the parameters and/or by appropriate choice of stationary phases, conditions of the organic modifier, and mobile phase pH on both separation dimensions with different selectivities. Therefore, orthogonal separations make 2D separations successful.9 The orthogonality between the two dimensions of separation was evaluated by plotting each peptide hit per fraction in the first dimension separation as a function of its retention time in a second dimension separation (Figure 18).7 The fact that the peptides are evenly distributed over the whole. 29.

(43) map provides a clear indication of the high degree of orthogonality for SCX x RPLC and HILIC x RPLC. A few reports suggest that the orthogonality of the SCX x RPLC approach is less than ideal.8 It is known that the separation in SCX is directed by the charge on the peptide. Since the charge states of the tryptic peptides identified from SCX were mainly +2 and +3, the peptides cluster in a narrow retention window. The majority of peptides elute from the column early in the analysis, leaving a portion of separation space that is relatively devoid of peaks. Therefore the resolution of the SCX x RPLC approach is worse than expected. The fact that SCX fractionation can be easily coupled with a UV/Vis detector is a distinct advantage, since it permits the elution of peptides to be on-line monitored. It is feasible to pool the collected fractions with approximately equal amounts of peptide for each LC-MS/MS analysis. The chromatographic peak areas, as determined from UV absorbance values, indicate the amount of peptide at each time point. One to seven minutes were pooled for the first fraction, 8~9 minutes for the second fraction, after 10~14 minutes collected one fraction per minute, during 15~22 minutes and 29~38 minutes one fraction every two minutes; after 39 minutes, the eluate was pooled into one fraction for every 15 minutes. The result showed that after this manipulation, fractionation based on peak area determined by UV absorbance permits more peptides to be identified. (Table 3) It has been reported that solution-IEF (pI based) gave a better focusing of peptides compared with SCX (based on charge).51 However, the improved peptide separation did not result in the detection of an increased number of peptides/proteins in this study. This could be attribute to a deterioration in. 30.

(44) recovery in the case of solution-IEF, although the manufacturer claims that the sample can be recovered from the gel in 95% yield. In addition, the peptide is migrating in the gel without any monitoring device, which makes it difficult to predict the actual amount of peptide in each compartment of the IEF solution. Another problem is that the amounts of peptides vary greatly in different fractions from the solution-IEF. As seen in Figure 16B, there are two gaps in the peptide distribution map. These are due to a lack of peptides with a specific pI value.17 Alkaline-RP provides good orthogonality (Figure 18E), because of the ionic nature of peptides, which made it possible to achieve a substantial separation orthogonality in the alkaline-RP x RP LC system using a high pH buffer in the first dimension and a low pH buffer in the second separation dimension, even when the column was packed with an identical sorbent.20 Charge, GRAVY and pI Value Distribution of Peptides Figure 17 shows the charge distribution of peptides identified in SCX. It is known that singly and doubly charged peptides elute at the very beginning of the separation, whereas peptides carrying three or more charges are retained on the column more strongly and elute at higher salt concentrations. As shown in Figure 17A, the majority of the peptides identified from SCX are doubly charged peptides, which are present in fractions 2 through 8. Triply charged peptides comprise a certain amount of the total peptides identified, and are mainly present in fractions 9 through 18. More charged peptides, in relatively low amounts, are present in the last five fractions, when the majority of triply charged peptides had already eluted from the column. GRAVY (grand average of hydropathy) for a peptide or protein is. 31.

(45) calculated as the sum of hydropathy values of all of the amino acids, divided by the number of residues in the sequence.59 For either acidic-RP or alkaline-RP separation peptides based on hydrophobicity, peptides with a weaker hydrophobicity tend to elute early in the analysis, while peptides with a stronger hydrophobicity elute at higher concentrations of organic modifier. In contrast with reverse phase, peptides with a stronger hydrophobicity elute from the HILIC column early in the process. The GRAVY distributions for acidic-RP and alkaline-RP have the same trend, the peptides identified in early fractions have smaller GRAVY values and peptides identified in later fractions have higher GRAVY values (Figure 19A). The GRAVY distribution for HILIC (Figure 19B) is contrary to RP, peptides in early fractions have larger GRAVY values and those in later fractions have smaller GRAVY values. HILIC also exhibited a fairly pronounced advantage in identifying peptides with lower GRAVY values, in comparison with alkaline-RP (Figure 20). The amount of peptides with a GRAVY value below -0.5 identified by HILIC was 2966, with 2150 being identified by alkaline-RP respectively. This clearly shows that the HILIC fractionation approach permitted more hydrophilic peptides to be identified. Solution-IEF utilizes differences in pI values for peptides as the basis for separation. A 24 cm long IPG gel strip with a linear pH gradient ranging from 3 to 10 was used in this experiment; the gel strip was divided into 24 sections and each section had a pI range of 0.29. The pI value for the distribution of peptides identified by solution-IEF is demonstrated in Figure 14F and Figure 14G. Peptides were unevenly distributed along the IPG strip scale; there are two gaps in the map; one in the pI range 4.45 to 5.03 and the other in the pI. 32.

(46) range 7.64 to 8.51. The gap of peptides in certain fractions could be attributed to a lack of possible amino acid combinations that could be present in peptides with adequate pI values.56 There are several reports indicating that the distribution of a proteome digest over the OFFGEL IPG strip is not dependent on the sample nature or the specific organism (eukaryote or prokaryote).17, 56, 60 Complementarity of SCX x RPLC, HILIC x RPLC, Alkaline-RP x RPLC and sIEF x RPLC Methods In order to systematically compare the relative efficiencies of 2D separations for complex protein mixtures, a consistent peptide load in the second dimension, a consistent number of collected fractions (24), consistent LC-MS/MS conditions, and consistent database search parameters were employed. Table 3 summarizes the total proteins/peptides identified by Mascot for each workflow. As shown in Table 3, SCX x RPLC using desalted samples gave the greatest number of proteins identified, followed closely by HILIC x RPLC. The alkaline-RP x RPLC and solution-IEF x RPLC gave average results while the RP x RPLC gave the worst results (as a control). To evaluate the overlap between the four methods, Venn diagrams, based on the comparison of identified proteins and peptides across the different methods, were constructed using Scaffold (Figure 21). A total of 1762 proteins were identified within all sets of methods, SCX covered 96.54% of the total proteins identified (Figure 21A). When SCX and HILIC were used, 98.92% of the total proteins were identified. At the peptide level (Figure 21B), SCX and HILIC covered 70.04% and 58.75% of the total peptides identified, respectively, but the sets of peptides were somewhat independent of one another. The amount. 33.

(47) of peptides identified with all setups is rather high in comparison with the amount of proteins identified with all setups, which explains the fact that different peptides may contribute to the identification of the same protein. The combination of methods not only increases the marginal amount of identified protein but also increases the amount of identified unique peptides identified to a significant extent. Table 4 listed 10 proteins identified from our experiment. The peptide per protein ratio listed in the table was increased after combining the data from the four different fractionation approaches. For example, in the case of the early endosome antigen 1 (accession number: IPI00329536.2) , the sequence coverage was only about 19.99% in the SCX approach but after combining the data from the four fractionation approaches, a 33.1% sequence coverage was realized. This indicates that, although SCX x RPLC is capable of generating the largest numbers of peptide and protein identifications, the four fractionation methods complement each other to some degree. Table 5 lists the number of phosphopeptides identified from each fractionation method, SCX gave the best results (48) and solution-IEF gave the worst results (17). When the data from the four fractionation methods are combined, the number of unique phosphopeptides identified were substantially increased to 83 (73% higher than SCX x RPLC only). The increased amount of distinct peptides being identified is especially beneficial, not only for confidently identifying proteins but also for providing an enhanced feasibility in proteomic studies with post-translational modifications and peptide-based quantification approaches using stable isotope labeling, such as iTRAQ, SILAC, or TMT. For quantification approaches,. 34.

(48) improved sequence coverage of proteins will increase the number of peptide matches per protein, thus reducing false positive identifications and enabling statistical quantitation for a greater number of identified proteins.. 35.