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Quantitative proteomic approaches for studying phosphotyrosine signaling Shi-Jian Ding, Wei-Jun Qian and Richard D Smith†
Summary & conclusions
Protein tyrosine phosphorylation is a fundamental mechanism for controlling many aspects of cellular processes, as well as aspects of human health and diseases. Compared with phosphoserine and phosphothreonine, phosphotyrosine signaling is more tightly regulated, but often more challenging to characterize, due to significantly lower levels of tyrosine phosphorylation (i.e., a relative abundance of 1800:200:1 was estimated for phosphoserine/phosphothreonine/phosphotyrosine in vertebrate cells). In this review, we outline recent advances in analytical methodologies for enrichment, identification and accurate quantitation of tyrosine-phosphorylated proteins and peptides. Advances in antibody-based technologies, capillary liquid chromatography coupled with mass spectrometry, and various stable isotope labeling strategies are discussed, as well as non-mass spectrometry-based methods, such as those using protein/peptide arrays. As a result of these advances, powerful tools now have the power to crack signal transduction codes at the system level, and provide a basis for discovering novel drug targets for human diseases.
Expert commentary
Expert Rev. Proteomics 4(1), 13–23 (2007)
Five-year view
Tyrosine phosphorylation is a central regulatory mechanism in cell signaling, and many of the signaling components involved in this mechanism are highly conserved among the eukaryotic systems [1–3]. Compared with phosphoserine (pSer) and phosphothreonine (pThr), phosphotyrosine (pTyr) signaling is more tightly regulated, but often more challenging to characterize, due to significantly lower level of tyrosine phosphorylation (i.e., a relative abundance of 1800:200:1 was estimated for pSer/pThr/pTyr in vertebrate cells [4]). It is generally accepted that tyrosine phosphorylation is coordinately regulated by the balanced action of protein tyrosine kinases and protein tyrosine phosphatases. In the human genome, there are only 90 known genes that code for protein tyrosine kinases (of which, 85 are thought to be catalytically active) [5], and, to date, 107 protein tyrosine phosphatases genes have been identified (81 of which are active with the ability to dephosphorylate pTyr) [6]. Abnormal regulation of tyrosine phosphorylation plays a role in the
CONTENTS Enrichment strategies for phosphotyrosine analysis Detection & identification of phosphotyrosine proteins & peptides Quantitative analysis of phosphotyrosine signaling
Key issues References Affiliations
†
Author for correspondence Pacific Northwest National Laboratory, Biological Science Division & Environmental Molecular Sciences Laboratory, PO Box 999, MSIN: K8-98, Richland, WA 99352, USA Tel.: +1 509 376 0723 Fax: +1 509 376 7722
[email protected] KEYWORDS: cell signaling, immunoprecipitation, mass spectrometry, proteomics, stable isotope labeling, tyrosine phosphorylation
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10.1586/14789450.4.1.13
pathogenesis of numerous human diseases, which range from cancer to immune deficiencies [2]. The fact that more than half of human tyrosine kinases have been reported as oncogenes further illustrates the vital role of tyrosine phosphorylation in human cancer [7]. As a result, protein tyrosine kinases have become the major targets for many drug-discovery programs. The best known examples include tyrosine kinase inhibitors directed against BCR-ABL in leukemia and the HER2/ErbB2 receptor in breast cancer [8]. Herceptin® (trastuzumab, a monoclonal antibody that targets the HER2 receptor, was approved in 2000 for treating advanced breast cancer, and Gleevec® (imatinib), a small-molecule inhibitor of the ABL tyrosine kinase, was approved a year later for treating chronic myelogenous leukemia. The success of these specific tyrosine kinase inhibitors validates the clinical relevance of pTyr signaling studies. Despite the biological significance of tyrosine phosphorylation, analyzing pTyr signaling has been technologically challenging for several
© 2007 Future Drugs Ltd
ISSN 1478-9450
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reasons. First, signaling components involved in tyrosine phosphorylation are present in extremely low abundance within the cells (i.e., only a few hundreds copies per cell) [2]. Second, tyrosine phosphorylation typically presents at relatively low stoichiometry for a given protein, which is similar to other post-translational protein modifications. pTyr represents only 0.05% of all phosphorylated amino acid content, and typically less than 1% of the total identified peptides from a proteome analysis are phosphorylated or post-translationally modified [9]. Third, tyrosine phosphorylation is reversible, and thus, phosphorylated residues within proteins can be rapidly dephosphorylated once the stimulus has been removed. For this reason, it is important to take precautions during sample processing to inhibit phosphatase activity. Overcoming these analytical challenges requires reliable technologies that provide both the high specificity and sensitivity for quantitative analysis of tyrosine phosphorylation. Over the past several years, significant technological advances have been made in the areas of enrichment, detection, and quantitation. As a result of the increased analytical power afforded by these advances, new opportunities are available to those seeking to gain a greater understanding of the pTyr-dependent cell signaling mechanism at the system level. Enrichment strategies for phosphotyrosine analysis
An enrichment strategy to selectively isolate pTyr is generally a prerequisite for effective analysis, due to the extremely low abundance of pTyr proteins and peptides. FIGURE 1 depicts an overview of currently available pTyr enrichment strategies for various methods of detection. These different strategies
encompass antibody-based enrichment, PTB domain-based profiling, immobilized metal-ion affinity chromatography (IMAC), chemical derivatization approaches and other separation methods. Often, a combination of multiple approaches must be used to attain the enrichment specificity necessary for effective detection. pTyr antibody-based enrichment
Analysis of tyrosine phosphorylation by mass spectrometry (MS) has been greatly facilitated by the development of pTyrspecific antibodies, which can be used to immunoprecipitate tyrosine-phosphorylated proteins from whole cell lysate or to selectively enrich pTyr-containing peptides from complex peptide mixtures. At present, there are hundreds of different commercially available pTyr antibody products. A number of these products, such as 4G10, pY20, pTyr100, pY99 and pT66, have been successfully applied to enrich of pTyr proteins and peptides for MS analysis [10–14]. For global studies, the antibody should ideally bind to all pTyr proteins with equal or similar specificities; however, a particular antibody is often biased towards its specific cellular targets. Therefore, a common practice is to combine two or more different pTyr antibodies to improve pull-down coverage. For example, Zheng and coworkers used a mixture of pT66 and 4G10 combined with IMAC to investigate the interferon α-induced pTyr changes in Jurkat cells [14]. In another study, Blagoev and coworkers used a mixture of pTyr100 and 4G10 to purify and identify tyrosine-phosphorylated proteins and their binding partners in a time-course study of their activation upon epidermal growth factor (EGF) stimulation [10]. To date, most
Cell lysate Anti-pTyr or SH2 domain IP
Trypsin digestion
pTyr-enriched proteins
Peptide mixtures Trypsin digestion
1D/2D gel
Trypsin digestion
LC/MS/MS or MALDI-TOF
Anti-pTyr IP
Peptide mixtures
SCX
IMAC
LC/MS/MS
pTyr-enriched peptides
IMAC
Chemical modification
LC/MS/MS
Figure 1. Overview of techniques for enrichment and analysis of pTyr-containing proteins or peptides using mass spectrometry-based detection methods. IMAC: Immobilized metal-ion affinity chromatography; IP: Immunoprecipitation; LC: Liquid chromatography; MS/MS: Tandem mass spectrometry; pTyr: Phosphotyrosine; SCX: Strong-cation exchange; SH2: Src homology 2; TOF: Time-of-flight.
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Expert Rev. Proteomics 4(1), (2007)
Quantitative proteomic approaches for studying phosphotyrosine signaling
studies have applied pTyr antibodies for immunoprecipitation (IP) at the protein level rather than the peptide level. One of the advantages of pTyr enrichment at the protein level is that both nonphosphorylated peptides and phosphorylated peptides derived from targeted proteins can be identified by MS analysis, which improves the sequence coverage of the identified pTyr protein. In addition, pTyr protein binding partners can potentially be identified by a protein IP approach and these binding partners reflect phosphorylation-dependent protein–protein interactions that may be as equally important as tyrosine phosphorylation; however, this approach cannot distinguish between pTyr proteins or binding partners without identification and quantitation of the corresponding pTyr sites. Results from quantitative analysis can be used to discriminate the true binding partners from the nonspecific bindings [15]. The effective identification of low-abundance pTyr peptides is often difficult for protein-level IP experiments because most of the resulting peptides are nonphosphorylated peptides, originating from phosphoproteins and other specific or nonspecifically bound proteins. To address this challenge, Rush and coworkers recently reported an alternative strategy involving directly immunoprecipitating pTyr peptides from digested cellular protein extracts by using pTyr-specific antibody pTyr100 coupled noncovalently to protein G agarose [16]. Following standard liquid chromatography (LC) coupled with tandem MS (MS/MS) analyses, 688 pTyr-containing peptides and 628 pTyr sites from three distinct cell types were identified [16]. Several recent studies also have applied this direct peptide-level enrichment strategy to profile pTyr sites in growth factor signaling networks [17,18]. While the site-specific monitoring of protein tyrosine phosphorylation provides more explicit details regarding the regulation of proteins within the network, this approach precludes the analysis of other potential nonphosphorylated protein binding partners associated with tyrosine-phosphorylated proteins. pTyr binding domain-based profiling
An alternative to pTyr antibodies, phosphoprotein binding motifs such as Src homology (SH)2 domain, pTyr binding (PTB) domain and C2 domain of protein kinase C can be used as baits for purifying tyrosine-phosphorylated proteins and their interacting partners from cellular lysates [19–22]. SH2 domains are protein modules that recognize short phosphopeptide motifs composed of pTyr followed by three to five C-terminal residues, such as those generated by autophosphorylation of activated receptor tyrosine kinases [23]. The recombinant domain-containing proteins can be easily produced in bacteria, and bind well to their tyrosine-phosphorylated ligands in vitro. These properties suggest that SH2 domains are applicable for probing the tyrosine phosphorylation state of cellular protein samples. Blagoev and coworkers reported on the use of the SH2 domain of the adapter protein Grb2 as a bait for directly purifying EFG receptor (EGFR) and Src-interacting proteins from whole cell lysates [15]. The Grb2 protein specifically binds
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phosphorylated EGFR and Shc proteins. A total of 228 tyrosinephosphorylated proteins were identified and quantified from EGF-stimulated cells, of which 28 were enriched upon EGF stimulation [15]. Similarly, other PTB domains, such as PTB and C2, may also be used to functionally purify specific phosphorylated proteins in complex mixtures, although such applications have not been coupled to MS for tyrosine-phosphorylation identification to date. Global phosphopeptide-enrichment methodologies
In addition, to the immunoaffinity-based approaches, various other methodologies have been developed to globally enrich phosphopeptides in complex mixtures. Since these global methods do not specifically target pTyr, they are often used as secondary enrichment steps following pTyr IP enrichments. One of the most popular global methods is IMAC, which is based on the affinity of negatively charged phosphate groups for positively charged metal ions (e.g., Fe3+, Ga3+ or Ti4+) immobilized on a chromatographic support. The specificity of IMAC is significantly improved by peptide methylation prior to the enrichment [24]. Salomon and coworkers first reported the use of pTyr protein IP coupled with IMAC, which led to the identification of 64 pTyr sites on 32 proteins from human T cells and chronic myelogenous leukemia cells treated with imatinib [25]. Several recent applications also coupled the direct peptide-level pTyr IP strategy with IMAC to attain more effective identification of pTyr peptides [17,18]. Alternatively, various chemical derivatization methods have been developed that use chemical modification of the phosphate moiety as a means to enrich phosphopeptides; however, most of these methods involve hydroxide-mediated β-elimination that is only applicable to pSer and pThr peptide enrichment [26–28]. Two methodologies developed by Aebersold and coworkers can be applied for enriching both pSer/pThr and pTyr enrichments [29,30]. The first method reported by Zhou and coworkers utilizes a reversible carbodiimide-catalyzed condensation reaction to attach cystamine to a phosphate moiety, which enables phosphopeptides to be purified on glass beads that contain immobilized iodoacetyl groups [30]. However, the substantial sample loss, which is a consequence of the multiple protection and release, limits the applicability of this method to complex mixtures. More recently, Tao and coworkers from the same group presented a new chemical derivatization strategy for enriching phosphopeptides using dendrimer as a soluble polymer support [29]. Mixtures of peptides are first converted to the corresponding methyl esters to protect the carboxylate groups from further reactions during subsequent steps. The phosphorylated peptides are then covalently conjugated to the dendrimer support in a single-step reaction catalyzed by carbodiimide and imidazole. Modified phosphopeptides are released from the dendrimers via acid hydrolysis. When coupled to an initial anti-pTyr protein IP step, this method enabled a total of 97 tyrosine phosphoproteins and 75 pTyr sites in pervanadate-treated human T cells. While similar to the method reported earlier by the same group, the
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improvement gained by chemical derivatization stems primarily from the very simplified isolating procedure that employs a dendrimer-based soluble polymer support. Detection & identification of phosphotyrosine proteins & peptides
Following enrichment of pTyr proteins/peptides, various analytical technologies (gel electrophoresis, LC/MS/MS and different types of microarray approaches) have been applied to separate, detect and identify pTyr peptides/proteins. LC/MS/MS has recently established itself as a powerful tool; enriched phosphopeptides and phosphoproteins can be either separated by a 1D reverse-phase capillary LC or 2D-LC separation (e.g., strong-cation exchange coupled to reverse-phase LC) followed by MS/MS analysis for identification [31]. The overall performance of the LC/MS/MS platform depends significantly on the peak capacity of the online gradient reversephase capillary LC separations, the dynamic range of the MS system and the overall sensitivity. Due to the extremely low abundance of pTyr signaling proteins within the cells, the overall sensitivity of the LC/MS/MS analysis is particularly crucial for successful analyses. The LC/MS/MS sensitivity is largely dependent on the electrospray ionization efficiency and the overall MS performance. Significant improvements in sensitivity and separation efficiency have been achieved by employing smaller diameter capillary columns (inner diameter 10,000 ϕ) and small (
