4.1. Protein separations
4.1.1. General protein separation: chromatography and electrophoresis
Chromatography and electrophoresis have been used for cen-turies as a means of protein separation. Due to many diverse properties of samples, many techniques have been developed taking advantage of differences in chemistry, biology, size, shape, charge, hydrophobicity and biochemistry of the molecu-lar to separate the molecumolecu-lar forms found in biological samples [35]. Chromatography is usually used to separate different com-pounds in a mixture and to determine the exact amount of each compound. It is a powerful analytical technique because it can be used both qualitative and quantitative. Many types of available matrix used for column chromatography are usually packed in the column in the form of small beads and provided the different protein profiles of each separation. Affinity chromatography is usually used for purifying the target protein with high affinity binding upon the matrix used; for example, a molecule of anti-body or enzyme substrate directed interact a specific protein is attached to the bead. Otherwise, the lectin-agarose affinity chro-matography based on lectin specificity on carbohydrate moiety is also used to bind specific sugars for depletion of carbohy-drate moieties or enrichment of glycoproteins prior to proteomic analysis [36]. Gel filtration chromatography is used to sepa-rate proteins or peptides on the basis of size. Smaller molecules diffuse further into the pores of the beads and therefore move through the bed more slowly, while larger molecules enter less or not at all and thus move through the bed more quickly. Both molecular weight and three-dimensional (3D) shapes of proteins contribute to the degree of retention. Gel filtration chromatogra-phy can also be used for analysis of molecular size, separation of target protein in a mixture, and salt removal or buffer exchange from a preparation of marcromolecules. Ion-exchange chro-matography relied on charge–charge interactions between the proteins in sample and the charges immobilized on the resin can be subdivided into cation- and anion-exchange chromatogra-phy. Cation-exchange chromatography binds positively charged ions, while anion-exchange chromatography binds negatively charged ions. However, the utility of chromatography meth-ods as the sole separation technique are used to isolate proteins before mass spectrometry analysis.
Electrophoresis is a method that separates macromolecules either nucleic acids or proteins on the basis of size, electric charge, and other physical properties. Separation of charged
Fig. 4. Deglycosylation of glycoproteins in normal human serum by chemical method, (A) SDS-PAGE gel of deglycosylated proteins in serum samples. (M: protein markers; 1, normal human serum (NS); 2, NS + HF (anhydrous); 3, NS + HF (48–51%); 4, NS + TFMS; 5, ovalbumin (O); 6, O + HF (anhydrous); 7, O + HF (48–51%);
8, O + TFMS). (B) 2DE gel images of normal human serum before (B1) and after HF deglycosylation (B2).
molecules is based on their migration in an applied electric field. For instance, polyacrylamide gel electrophoresis (PAGE), since the pores in a PAGE gel are excluded the size of pro-teins, molecular sieving contributes to the resolving power of PAGE. Consequently, PAGE is a high-resolution method and one of the best available for separating complex mixtures of proteins, which require a small gel pore size for retardation.
High throughput technique has been developed which utilize protein separation by 1D or 2DE. The 2DE analysis of proteins is currently the highest-resolution analytical technique avail-able for the study of protein expression pattern and capavail-able of resolving thousands of protein in one experiment. The promi-nent point of 2DE as a separation technique is the orthogonality of the two-dimensional separation, based on vertically pI and horizontally molecular weight. The procedure has become the core technology to visualize the global change in protein expres-sion for proteome analysis with subsequent protein identification by mass spectrometry [37,38]. Since the high sensitivity and throughput of mass spectrometry were the main characteris-tic that provided the best methodology to identify protein of interest, the mass spectrometry has been widely recognized as a cornerstone of proteomic research.
4.1.2. Multidimensional proteomic approach
Many proteomic approaches have been attempted to increase the overall resolution of protein separation by combining
differ-ent techniques. Up-to-date, the first approach is still 2DE that used to characterize the complex protein mixtures, followed by trypsin proteolysis of visible proteins spots, and subse-quent analyzed the individual digested peptide by MALDI-MS [39–41]. Although the 2DE provides excellent resolving power, the dynamic range of this technique is still limited for detection of low abundant proteins. Some technical limitations are differ-ently in accomplishing reproducible separation and identifying separated proteins. Thus, the separation of such large number of component is not possible by only a single chromatographic or electrophoretic run[42,43]. The use of several independent dimensions significantly increases resolution of a separation.
Therefore, the combination of two or more orthogonal separation procedures dramatically improves the results in a larger number of protein or peptide being identified from complex proteome digests.
Alternative ways of multidimensional approaches employing liquid chromatography (LC) can potentially overcome some of the limitation of 2DE in proteomic analysis and are proposed as a way to separate protein and peptide with development of highly effective methods for peptide separation. The most current liquid phase separation methods can be achieved by using high performance liquid chromatography (HPLC), capil-lary isoelectric focusing (CIEF) and/or capilcapil-lary electrophoresis (CE). The protein analysis can combine two different separa-tion processes, chromatography or electrophoresis [40,44,45].
Fig. 5. 2DE patterns of pooled urine samples from normal (A) and lung cancer (B) by using different techniques of sample preparation. The urine samples were prepared by using ultrafiltration (A1, B1); acetone precipitation (A2, B2);
ACN/TFA precipitation (A3, B3); methanol/chloroform/water precipitation (A4, B4) and TCA in acetone precipitation, respectively.
For example, the protein analysis can use ion-exchange HPLC followed by reverse phase-HPLC (RP-HPLC) followed by CE or CIEF, and can also be coupled with different detection sys-tems, such as 2DE, MS or laser-induced fluorescence (LIF), to enhance the detectability and identification. Recently, a novel multiplexed microcapillary liquid chromatography system has been developed for automated and high throughput separation of complex protein/peptides sample in RP-LC subsequently fol-lowed by MALDI analysis[44]. This strategy allows a four-fold increase in sample throughput and relies on both MS and MS/MS analysis for quantitative and qualitative analysis of protein
mix-tures. The combination of liquid chromatography with different separation principle makes multidimensional chromatography more attractive technology. In addition, we also used the mul-tidimensional chromatographic methods using RP-HPLC and 2DE to separate the protein component of Naja naja kaouthia venom (Fig. 6). The 2DE images of the three RP-HPLC peaks demonstrated broad distribution of molecular weights and pI values. The 2DE result of one peak from the RP-HPLC elution contained many protein components that include protein aggre-gates, isoforms, or protein–protein interactions having the same hydrophobic property. Many trains of spots are presumed to be protein isoforms, due to post-translational modifications, as well as homologues with similar amino acid compositions. Using multidimensional chromatographic methods to prefractionate and analyze the snake venom proteome, the obtained protein patterns, protein identifications, and unique markers are very important and useful for further diagnostic and pharmaceutical applications.
4.2. Protein detection and quantification
The differential protein expression on gel electrophoresis including 1D and 2DE is analyzed by the shape, size, and inten-sity of the corresponding band or spot of proteins. The first stage in protein quantification is image acquisition and the method used depending on how the proteins were stained. The most popular and widely used methods of protein staining that are applied to reveal all the protein bands or spots are Coomassie colloid solution[46], Silver nitrate[47,48]and SYPRO®Ruby [49]. Coomassie and silver gel stains can be scanned with charge-coupled device (CCD) camera, while SYPRO®Ruby gel stain or fluorescently labeled proteins must be scanned using fluorescent image scanner, such as Typhoon 9200 series scanner (Amersham Bioscienes). Two dimensional difference in-gel electrophoresis (2D DIGE) is a relatively new technique in 2DE for multiplex quantitative analysis of the component proteins of related but different protein samples[50,51]. This technique allows label-ing protein mixtures with different fluorescent cyanine dyes, such as Cy2, Cy3 and Cy5 maleimides. These CyDyes are struc-turally similar, but spectrally different (Cy2,λem= 520 nm; Cy3, λem= 580 nm; Cy5,λem= 670 nm) fluorophors undergo nucle-ophilic substitution reaction with the thiol group of cysteine residues of proteins via a thioether linkage. The 2DE image shows the different protein spots with different fluorescent col-ors of labeled CyDyes on one gel image and the fluorescent intensity can be compared to allow quantification of each pro-tein spot. In our study, we employed 2D DIGE to investigate differentially expressed proteins in rice samples. For each of sample, Cy3-labeled proteins from CNT1 rice sample were com-bined with Cy5-labeled proteins from KMDML105 rice sample and separated by 2DE analysis (Fig. 7). Special image analysis software can be used to match the images, to quantitative the spots, to normalize the signals, and to provide the difference of expression of any set of two proteins by comparison. Com-pared with conventional 2DE, the co-migration of proteins on 2D DIGE can generate reproducible data and has the potential for high-throughput analysis.
Fig. 6. Multidimensional chromatography of Naja naja kaouthia venom using RP-HPLC and 2DE analysis. (A) RP-HPLC fractionation of N. naja kaouthia; (B), (C), and (D) are 2DE images of fractionation of peak area eluted from RP-HPLC; and (E) 2DE image of whole proteins from N. naja kaouthia venom.
In addition, the post-translational modifications of phospho-proteins and glycophospho-proteins can be directly detected by different staining methods[52–55]. Pro-Q Diamond phosphoprotein stain is a phosphoprotein specific fluorescence-dye that is available from Molecular Probes, and can be used to detect phospho-rylated tyrosine, serine, or threonine residues of proteins on SDS-PAGE and 2DE [53]. Otherwise, a recently developed approach for the detection of glycosylated proteins relies upon the utilization of a fluorescent hydrazide. Pro-Q Emerald 488 glycoprotein gel stain provides an attractive alternative to the labeling with radioactive sugars that conjugated to
glycopro-tein by periodic acid Schiff’s mechanism to use for specific glycoprotein detection[56]. Gels stained with both Pro-Q Dia-mond phosphoprotein stain and Pro-Q Emerald 488 glycoprotein stain can also be post-stained with SYPRO®Ruby dye, which allows sequential detection of total protein profile in the same gel. Furthermore, several labeling strategies have been devel-oping, based on the metabolic labeling for incorporation of a light or heavy form to the different experimental protein sample.
Recently, the stable isotope labeling is used for quantification of proteins[57,58]. One of most popular methods for isotope labeling is ICATs (isotope coded affinity tagging), based on two
Fig. 7. Separation of proteins in CNT1 and KDML105 rice samples on 2D difference in-gel electrophoresis (2D DIGE). Equal volumes of the two fluorescent dye-labeled samples were mixed, separated on 2DE by isoelectric focusing (IEF) followed by SDS-PAGE. The IEF was performed in an 18 cm Immobiline DryStrip with a pH range of 3–10 NL. The protein were further separated by SDS-PAGE on a 12.5% polyacrylamide gel, and scanned using the Typhoon 9200 series imager.
(A) Comparative protein expression profiling in CNT1 and KDML105 rice samples by 2D DIGE. (B) The CNT1 proteins were labeled with Cy3 (green color). (C) The KDML105 proteins were labeled with Cy5 (red color).
principles: (i) a short sequence of contiguous amino acids con-tains a sufficient information to identify that unique protein, and (ii) pairs of peptides tagged with the light and heavy ICAT reagents are chemically identical and therefore serve as ideal mutual internal standards for accurate quantification [59,60].
Another isotope tagging method is stable isotope labeling with amino acids in cell culture (SILAC) that has also widely used method to identify and quantitate complex protein samples[61].
Using SILAC approach involves growing cells two different bio-logical condition on normal and stable-isotope labeled media that supplemented with light or heavy isotope containing amino acids, the isotope labeled samples are then combined in equal ratios and subsequent analyzed quantification of proteins or peptides by MS. These labeling strategies employ mass spec-trometry to obtain quantitative information, which can determine the relative abundance for each peptide-pair. Any peptide-pairs
that are significantly different can be further sequenced using MS/MS and the relative amounts of the isotopic peaks can be determined on the basis of the intensities of the light and heavy peptides. It allows comparison samples to be combined and treated as a single sample throughout subsequent purification and analyses[61–63]. Although the strength of these techniques lies in its ability to allow quantification and identification within a single analysis, there is some limitation of each technique. In one limitation of isotopic labeling techniques, SILAC requires no chemical labeling or affinity purification steps because it relies on the normal biosynthetic machinery of cells, whereas ICAT uses a chemical, only cysteine containing peptides are retrieved non-specific binding to stable isotope mass tags. Thus, the strategies for protein quantification in proteomics depend on the use of general staining or labeling of particular classes of proteins, which is a significant component of proteomics.
4.3. Protein identification by mass spectrometry
Mass spectrometry has become an important tool for protein identification, peptide sequencing, identification and location of post-translational modifications of proteins[64–66]. In gen-eral, the mass spectrometer can be thought of as two distinct components of the ionization source and the mass analyzer.
The ionization source is the region of the instrument in which the sample of interest is ionized, with a positive or negative charge, and then desorbed into the gas phase. The mass ana-lyzer is where the gas phase ions created in the source region are guided through the instrument to the detector, where their mass-to-charge (m/z) ration is measured. Two ionization sources of electrospray ionization (ESI) and matrix-assisted laser des-orption/ionization (MALDI) are currently the principal methods for peptide/protein ionization. ESI is the choice of identifica-tion method for proteins, oligonucleotides and metal complexes, which can produce molecular ions directly from samples in solution and transfer into the gas phase. However, the effi-cient of ionization is directly impacted by the solution phase chemistry of the various peptides that varies in accordance to their physicochemical properties, including pKa value, polar-ity, hydrophobic or hydrophilic index and ionization potential, and by the concentration and type of peptides infused into the ionization source. MALDI coupled with time-of-flight (TOF), known as MALDI-TOF, has been developed for the ionization of relatively large polypeptides and proteins and its application has widened to incorporate glycoproteins, oligonucleotides and complex carbohydrate[67,68]. It is used predominantly for the analysis of simple peptide mixtures, such as the peptide obtained from an interest of single spot that separated on 2DE. Also, it has been used to analyze the large m/z range mass used for protein identification by means of peptide fingerprinting but it was suitable for analysis of material obtained from organisms with known complete nucleotide sequence of genome[69–71].
However, MALDI-TOF still has a limitation in the analysis of low molecular mass proteins that delivered few peptides, and the identification is often based on a low number of matches.
Although identification of small proteins by MALDI-TOF is not efficient, the combination of MS/MS technologies, such as TOF/TOF, hybrid quadrupole time-of-flight (Qq-TOF), or LC–MS/MS, may be useful and more advantageous for peptide sequencing[72–74].