One of the most significant biological achievements to emerge during the past 40 years has been the completion of draft DNA sequences of the human genome.

Chapter 1 – Introduction

1-1 Phosphoproteomics

1-1.1 From Genomics to Proteomics

One of the most significant biological achievements to emerge during the past 40 years has been the completion of draft DNA sequences of the human genome.

The International Human Genome Sequencing Consortium, led in the United States by the National Human Genome Research Institute and the Department of Energy, published its scientific description of the finished human genome sequence, reducing the estimated number of human protein-coding genes from 35,000 to only 20,000-25,000, a surprisingly low number for our species.

Although the number is surprising low, a gene can be transcribed to pre-mRNA that may be processed to one mRNA or by alternative splicing to several forms of mRNAs. The transcripts are translated into functional proteins - the ultimate operating molecules producing the physiological state.

Several processes in RNA levels and post-translational modifications of proteins would

add large amounts of additional diversity to the expression profiles of gene products.

The term “proteome” was first introduced to describe the set of proteins coined in 1994

as a linguistic equivalent to the concept of genome.

In contrast to the “static” genome

sequence, the proteome is a highly dynamic entity due to their abundance, state of

modification, subcellular location, etc. depends on their physiological state of the cell

or tissue.

The study of the proteome, called proteomics, initially was used to

describe the study of the expressed proteins of a genome using 2D gel electrophoresis.

This approach is now referred to as “expression” proteomics. The scope of proteomics now is used to describe the complete set of proteins that is expressed, and modified following expression, by the entire genome in the lifetime of a cell. It is also used in a less universal sense to describe the complement of proteins expressed by a cell at any one time. Today, proteomics is scientific discipline that promises to bridge the gap between our understanding of genome sequence and cellular behavior, and it can be viewed as more of a biological assay for determining gene function.

1-1.2 Post Translational Modification

In addition to the protein expression, the posttranslational modification of the protein determinates protein turnover, localization, activity or binding interactions.

The huge majority of eukaryotic proteins are posttranslational modified and more than 200 posttranslational modifications of amino acids have been reported thus far. These modifications act on individual residues either by cleavage at specific points, deletions, additions or having the side chains converted or modified. Post translational modification may involve the formation of disulfide bridges or attachment of any of a number of biochemical functional groups, such as acetate, phosphate, various lipids and carbohydrates. Some posttranslational modifications extend the range of possible functions a protein by introducing other chemical groups into the makeup of a protein.

Such chemical changes may alter the hydrophobicity of a protein and thus determine if the modified protein is cytosolic or membrane-bound.

1-1.3 Protein Phosphorylation

For the wide variety of posttranslational modifications, the protein phosphorylation is most common, about one third of the human proteins contain covalently bound phosphate, and five-hundred protein kinases and a third that number of protein phosphatases encode by human genome. The phosphorylation adds two negative charges to a protein and allows the formation of hydrogen bonds, which leads to changes in electrostatic interactions, substrate binding, conformation, and catalytic activity, as is important for biological function. The phosphorylation regulates protein function and localization and involves in ubiquitous regulatory mechanism in both eukaryotes and prokaryotes.

Intracellular phosphorylation is regulated by protein kinases, which are activated in response to extracellular ways, kinase cascade activation, membrane transport, gene transcription, and motor mechanisms. The study of cell biology is now littered with examples of regulation by phosphorylation:

increasing or decreasing the biological activity of an enzyme, helping move proteins

between subcellular compartments, allowing interactions between proteins to occur, as

well as labeling proteins for degradation. Several diseases have been recognized to be

associated with the abnormal phosphorylation of cellular proteins. For example, a

major virulence factor of Yersinia is protein tyrosine phosphpatase: this class of

bacteria causes several serious diseases, including the bubonic plague, which has been

responsible for many pandemics over past millennium.

These include the Black Death,

which killed 25 % of the population of Europe in the 12th an 13th centuries. This

phosphatase can enter human cells causing uncontrolled dephosphorylation of many

tyrosine residues that rapidly proves fatal. Therefore, phosphorylated proteins are

attractive drug targets. Large-scale identification of phosphorylated kinase substrates

will certainly enhance our understanding of diverse biological phenomena, potentially

leading to targeted intervention in any number of disease paradigms.

1-2 Analysis of Protein Phosphorylation

1-2.1 Analytical Challenges of Protein Phosphorylation

To gain further insight into regulation of cellular function by reversible phosphorylation, it is often necessary to characterize the phosphorylation state of specific proteins under certain conditions. However, global analysis of protein phosphorylation remains a major analytical challenge. Mann et al. have reviewed the detailed reasons for this.

First, the stoichiometry of phosphorylation is generally relatively low - only a small fraction of the available intracellular pool of a protein is phosphorylated at any given time as a result of a stimulus. Second, the phosphorylated sites on proteins might vary; implying that any given phosphoprotein is heterogeneous (i.e. it exists in several different phosphorylated forms). Third, many of the signaling molecules are present at low abundance within cells and, in these cases; enrichment is a prerequisite before analysis. Fourth, most analytical techniques used for studying protein phosphorylation have a limited dynamic range, which means that although major phosphorylation sites might be located easily, minor sites might be difficult to identify. Finally, phosphatases could dephosphorylate residues unless precautions are taken to inhibit their activity during preparation and purification steps of cell lysates.

Therefore, it needs that the high dynamic range of analytical techniques employed for the study of protein phosphorylation.

1-2.2 Analytical Techniques for the Protein Phosphorylation

There are several analytical techniques developed for the analysis of protein phosphorylation. The conventional approach for characterizing protein phosphorylation relies on

P labeling with 2D-PAGE quantification by scintillation counting or audioradiography.

The identification of protein and phosphorylation site can be achieved by using Edman sequencing.

However, these techniques require relatively long time for sample handling, and are insufficient when dealing with complex protein mixtures. In addition, for the method of

P labeling, it suffers from the difficulties associated with the use of radioactive isotopes. For the Edman sequencing, it requires large quantities of sample and also limited by the long analysis time. In recent years, mass spectrometry has become the choice for protein phosphorylation analysis. Table shown below provides a comparison of mass spectrometry-based approaches (mainly refer to LC-MS/MS) to methods using Edman sequencing and

P labeling.

Mass spectrometric method for identification and characterizing phosphoproteins is inherently better than the others.

32

P-labeling Edman sequencing

Mass spectrometry

Sensitivity

Radioactivity

Localization of phosphorylation sites

no

yes ( However, tyrosine phosphorylation sites can be difficult)

yes

Sample

throughput

Homogeneous

protein required

high 32

P-labeling Edman

sequencing

Mass spectrometry

Sensitivity

Radioactivity

Localization of phosphorylation sites

no

yes ( However, tyrosine phosphorylation sites can be difficult)

yes

Sample

throughput

Homogeneous

protein required

high

1-2.3 Mass Spectrometry Analysis of Protein Phosphorylation

The introduction of mass spectrometry has greatly advanced the analytical sciences, especially in biomolecular study. The soft ionization methods of ESI

and MALDI

allowed mass determination of large biomolecules. Those two important discoveries were honored with the Nobel Prize in Chemistry in 2002. Both ESI and MALDI mass spectrometry are amendable to phosphoprotein analysis, but most work to date has been performed by LC-MS/MS where peptide separation is combined with on-line ESI tandem mass spectrometry for automated peptide sequencing. MALDI mass spectrometry is a simple, sensitive and robust technology for peptide analysis, but it has lacked practical capabilities and sensitivity for peptide sequencing. MALDI mass spectrometry is applied to characterization of protein phosphorylation typically based on the 80 Da mass decreasing in a phosphopeptide mass after alkaline phosphatase treatment.

ESI is an attractive ionization method for characterization of phosphopeptides. Multiply protonated peptide ions fragment efficiently during LC-MS/MS by collision-induced dissociation (CID) is suitable for identification of the primary structure information which is extremely useful in verifying proposed phosphopeptides and in the determination of the phosphorylation sites within the peptide.

1-2.4 Challenges Associated with Mass Spectrometry Analysis

Although mass spectrometry is a sensitive and specific analytical method for

characterization of phosphoprotein, it is still an analytical challenge for several

reasons.

First, the ionization efficiency of phosphopeptides is strongly reduced when

compared with their nonphosphorylated counterparts, which is thought be the result of

increased hydrophilicity and acidity because of the phosphoryl group. Phosphopeptides

are generally detected with low efficiency by mass spectrometry, and this is referred to

as the suppression effect. Second, phosphorylated amino acid residues can interfere

with enzymatic cleavage behavior. The phosphoserine or phosphothreonine residues

from cleavage side reduce significantly the cleavage rate of trypsin. The reduced

cleavage efficiency results in larger peptides, which make MS analysis more

complicated. Third, phosphopeptides easily lose the phosphoryl group during mass

spectrometric experiments which especially come about for phosphoserine and

phosphothreonine. Fourth, phosphopeptides are often relatively hydrophilic and may

elute in the void volume when using RP-HPLC to separate. Therefore, to circumvent

these limitations, a number of purification and enrichment strategies for the protein

phosphorylation were developed to reduce the ionization suppression and increase the

detection limit of phosphopeptides.

1-3 Enrichment Strategies to Analyze Phosphopeptides

There are several affinity-based techniques used for the phosphopeptides/protein enrichment, including immunoprecipitation, chemical modification, immobilized metal affinity chromatography and strong cation exchange, which will be discussed in this section.

1-3.1 Phosphospecific Antibodies

Antibodies are routinely used to immunoprecipitate specific proteins. For tyrosine-phosphorylation, several excellent antibodies suitable for selective immunoprecipitation of tyrosine-phosphorylated proteins facilitate identification of tyrosine phosphorylated sites.

However, antibodies against phosphoserine and phosphothreonine have not been applied routinely because of the reduced efficiency.

Therefore, although these antibodies have been relatively effective at enriching and identifying low-abundance tyrosine phosphoproteins, they are not very good at enriching for all the phosphopeptides. Thus these proteins must be enriched using alternative methods.

1-3.2 Chemical Modification

Recently, several ways to identify and purify phosphopeptides have been based on

selective chemical modification of the phosphoryl moiety prior to analysis. This

usually makes use of a β-elimination reaction that occurs when phosphoserine and

phosphothreonine residues are exposed to strongly alkaline conditions.

The resulting dehydroalanine or dehydroaminobutyric acid residues can be detected using tandem mass spectrometry after chemical modification with ethanedithiol as a nucleophile, which provides a new reactive thiol group serving as linker for attachment of a biotinylated affinity tag.

However, an undesired side effect involving side chain on cysteine and methionine residues can occur. In addition, the main disadvantages of this chemical modification method are that it is not applicable to tyrosine phosphorylation and that the yield from the β-elimination reaction tends to be sub-stoichiometric.

Another problem is the requirement of multiple steps for chemical modifications and purification which will lead to substantial sample loss.

1-3.3 Immobilized Metal Affinity Chromatography

The immobilized metal affinity chromatography for the enrichment of

phosphopeptides exploits the high affinity of phosphoryl groups towards a

metal-chelated stationary phase, especially Fe

and Ga

.

The particularly strong

affinity between the metal ions and phosphate is due to metal coordination of two

oxygen atoms in the phosphoryl group. Because it is based on presence of negatively

charged phosphoryl groups, IMAC can be used to enrich for phosphorylated serine,

threonine, and tyrosine residues. Two types of metal-chelated stationary phases which

are iminodiacetic (IDA) and nitrilotriacetic acid (NTA) are coupled to a support, such

as Sepharose, agarose, or macroporous silica. Phosphopeptides are usually adsorbed at

acidic pH (2.5~5) with 0.1~1 % acetic acid, and eluted increasing pH. An increase in

pH induces hydroxyl ions to compete with bound phosphoryl groups. Another elution

can be performed by the addition of Mg

or phosphate ions; Mg

compete with the

immobilized metal ion for the phosphoryl groups and phosphate ions compete with the phosphoryl group on the peptides. IMAC columns have been used in combination with RP-HPLC and detect by mass spectrometry. Suppression effects can be greatly reduced by selectively enriching for phosphopeptides on metal ion affinity media prior to mass spectrometry analysis. However, a major issue is that the specificity of this procedure is variable because of affinity for acidic groups and electron donors. Phosphoryl groups are strongly coordinated and carboxyl groups are weakly coordinated by the metal ions.

Therefore, reduced IMAC efficiency occurred due to nonspecific binding of unphosphorylated peptides particularly containing high levels of glutamic and aspartic acid residues. Recenrly, Ficarro et al. attained a much higher specificity using esterification of acidic residues before IMAC enrichment, but sample recovery remains a potential limitation of this method.

The IMAC strategy is highly efficient, sensitive, and simple. With further refinement, this technique may offer the best opportunity for large-scale phosphorylation analysis.

1.3.4 Strong Cation Exchange

SCX is often used as a primary separation strategy for complex peptide mixtures

before analysis by reverse phase liquid chromatography, and separates peptide ions

based on solution charge state resulting from protonation/deprotonation of basic and

acidic groups. The most common implementation utilizes an on-line system in which

complex mixtures are adsorbed to a bi-phasic column packed with SCX and reverse

phase resins. Several salt “bumps” are then initialed to release discrete peptide

fractions to the reverse column for analysis. Although this strategy has been widely successful for the analysis of complex peptide mixtures, the ability of SCX chromatography to enrich phosphopeptides recently has been reported by Steven P.

Gygi et al.

1-4 Objectives of This Study

The identification of phosphorylation sites is most robustly accomplished by MS.

With tandem MS, phosphopeptides are fragmented to determine their sequence and to pinpoint the specific serine, threonine, or tyrosine. In this thesis, we aim to establish a large-scale identification platform for protein phosphorylation. The specific aims are as follows:

(1) Enrichment of phosphopeptide with IMAC chromatography. The parameters including column capacity, binding specificity, elution buffer and sample recovery were studied in details for optimal enrichment of phosphopeptides by IMAC column.

For the detection of low abundant phosphopeptides, the enrichment of phosphopeptides from crude, complex mixtures by IMAC is efficient and comparable to direct nLC-MS/MS analysis. The combination of IMAC with nLC-MS/MS permits good performance in detection and analysis of phosphopeptides, and has demonstrated great success for complex samples.

(2) Large-scale identification of phosphorylation sites in Jurket T cell.

Coupling of advanced separation methodologies were performed to reduce the sample complexity and extend the detection dynamic range.

Prior to IMAC chromatography and MS, advanced separation methodologies

provides powerful means to detect and analyze dynamic changes of low abundant

phosphoproteins in cells on subcellular level. In this study, using SDS-PAGE with

IMAC-nLC-MS/MS analysis, this approach has been used for determining large-scale

phosphorylation sites on human Jurkat T-cell. This robust technology platform can be

broadly applicable to profiling the dynamics of phosphorylation.