PDF Proteomics of Biological Systems: Protein Phosphorylation Using Mass Spectrometry Techniques

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Proteomics of Biological Systems: Protein Phosphorylation Using Mass Spectrometry Techniques. Author(s). Bryan M. Ham. First published
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Signaling pathways have also been discovered that amplify the initial signal; kinase A activates multiple kinases, which in turn activate additional kinases.


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With this type of signaling, a single molecule, such as a growth factor, can activate global cellular programs such as proliferation. Signal transduction cascades amplify the signal output. External and internal stimuli induce a wide range of cellular responses through a series of second messengers and enzymes. Linear signal transduction pathways yield the sequential activation of a discrete number of downstream effectors, while other stimuli elicit signal cascades that amplify the initial stimulus for large-scale or global cellular responses.

The intensity and duration of phosphorylation-dependent signaling is regulated by three mechanisms:. While dephosphorylation is the end goal of these two groups of phosphatases, they do it through separate mechanisms.

Phosphorylation | Thermo Fisher Scientific - US

Because of the influence that phosphorylation has on biological processes in general, a huge emphasis has been placed on understanding the biological role of protein phosphorylation in the context of human disease. Small-scale protein phosphorylation is commonly performed to study the activity of a small number of proteins, while phosphoproteomic analyses are increasingly used to understand the global dynamics of phosphorylation of entire protein families.

Current approaches to study protein phosphorylation include immunodetection, phosphoprotein or phosphopeptide enrichment, kinase activity assays and mass spectrometry. Because of the detrimental effect that phosphatases can have on the detection of phosphorylated proteins, broad-spectrum phosphatase inhibitors are commonly added to cell lysates in many phosphodetection strategies. Phospho-specific antibodies raised against specific phospho-epitopes on target proteins are a core tool for studying site-specific protein phosphorylation.

On a more global scale, antibodies have been developed to detect the phosphorylation of specific amino acids pS, pT, pY. Phospho-specific antibodies can be used for traditional western blotting, immunoprecipitation IP , immunohistochemistry IHC , ELISA, flow cytometry and, more recently, immobilization onto solid support arrays. Although phospho-specific antibodies are popular amongst the research community, only a small fraction of them are highly specific for their targets and many have issues with low sensitivity.

These assay kits are designed to deliver accurate, sensitive, and fast protein quantitation of total and phosphorylated, modified, or cleavage site-specific proteins in a broad range of sample types. Detection of p38 MAP protein. Key dynamic changes in protein phosphorylation occur on low abundance proteins, which require enrichment prior to proteomic analysis. MOAC is most commonly performed with TiO2-chelated resins to form bidentate complexes with phosphates, while IMAC employs Fe-chelated support to form tri- or tetradentate complexes with phosphates.

Commercial kits are also available that have proprietary phosphate-binding elements for phosphoprotein enrichment.

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Kinases ATPases and GTPases can also be enriched, although this approach does not provide information on the proteins that these enzymes modify. These enrichment strategies use nucleotide derivatives that bind to the active site of kinases or GTPases depending on the derivative used and mediate the covalent attachment of a modified biotin desthiobiotin. Because desthiobiotin exhibits reversible binding to streptavidin, labeled kinases or GTPases can be enriched from samples.

Specific enzymes can be enriched using consensus sequences of downstream proteins as probes. The following table provides information about the performance of the Thermo Scientific Pierce Phosphoprotein Enrichment Kit designed for the efficient enrichment of phosphorylated proteins derived from mammalian cells and tissues. Recent mass spectrometric strategies using stable isotope labeling by amino acids in cell culture SILAC or labeling of peptides in vitro with tandem mass tags have paved the way for the relative determination of changes in phosphorylation. Absolute quantitation strategies are also available using isotopically "heavy" peptide standards.

These approaches allow researchers to understand global phosphoproteomic changes in response to different stimuli or disease states. The diagram below illustrates the nature of complexity associated with protein sample preparation for MS. Phosphopeptide enrichment reduces sample complexity and is required prior to MS due to the low stoichiometry and poor ionization of phosphopeptides. Phosphoprotein enrichment is enhanced with the aid commercially available kits.

Protein availability for quantitative proteomic analysis is limited. Protein abundance and sample complexity are significant factors that affect the availability of proteins for mass spectrometric quantitation. Note: You clicked on an external link, which has been disabled in order to keep your shopping session open. Search Thermo Fisher Scientific. Search All. See Navigation. Page contents Mechanism of phosphorylation Protein kinases Signal transduction cascades Protein phosphatases Methods of detection Recommended reading.

Mechanism of phosphorylation. Protein Expression Handbook. Protein kinases. Signal transduction cascades. Protein phosphatases. Methods of detection. Immunodetection Phospho-specific antibodies raised against specific phospho-epitopes on target proteins are a core tool for studying site-specific protein phosphorylation.

Recommended reading. Cohen P The regulation of protein function by multisite phosphorylation--a 25 year update. Trends Biochem Sci — Yaffe MB Phosphotyrosine-binding domains in signal transduction. Nat Rev Mol Cell Biol — Bantscheff M et al. Anal Bioanal Chem — Manning G et al. Science — Walsh C Enzymatic reaction mechanisms. San Francisco CA : W.

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Walsh C Posttranslational modification of proteins: Expanding nature's inventory. The unwanted trypsin and keratin peaks were first removed using the Peakeraser software, which eliminated contaminated spectra from the total raw MS spectra of the DEPs Figure 3 and Table S1. The Peakeraser filter could partly eliminate the contamination peaks from the raw MS spectra, and further manual filter of the mistaken assignments of contaminated peptides to possible phosphopeptide largely diminished the prediction false-positive rate.


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  • Because some protein spots on 2-DE gels are not composed of single proteins but rather a mixture of multiple proteins [26] , mistaken assignments of peptides from co-migrated low abundance proteins to phosphopeptides of target proteins are possible when the Mw of the two are approximate. How to efficiently eliminate these false predictions? In MALDI-TOF mass spectra, phosphopeptide ions and the corresponding non-phosphopeptide ions always coexist and the former are always much weaker than the latter because the phosphopeptides are sub-stoichiometric and not prone to ionization [27].

    Based on this principle, the predicted phosphopeptides which do not coexist with their corresponding non-phosphopeptides or which are more intense than the corresponding non-phosphopeptides, are considered as false predictions. When the in silico run was ready, we thus further manually filtered the mistaken prediction through comparing the intensity of the predicted phosphopeptide ions and the corresponding non-phosphopeptide ions.

    Because 13 of the 48 identified phosphopeptides are actually repeatedly detected peptides for 5 phosphorylation sites Table 1 , there are total 40 unique phosphorylation sites identified.

    High Throughput Profiling of Serum Phosphoproteins/Peptides Using the SELDI-TOF-MS Platform

    B Percentage of phosphorylated amino acids among a total of 40 unambiguously identified phosphorylation sites. A great amount of information was obtained from the phosphorylation site identification results. Of the 40 identified unique phosphorylation sites, 15 were found to have been identified in other plant cells via BLAST searches of the 40 phosphopeptides in the P 3 DB database [21] , which means that the remaining 25 represent the first reports of new phosphorylation sites in plant cells Table 1.

    In addition, 22 of the 40 unique phosphorylation sites were found to be phosphorylated on serines, whereas 14 and 4 were phosphorylated on threonines and tyrosines, respectively Figure 4 -B. The pivotal significance of identifying the phosphorylation sites of the DEPs in elongating cotton fiber cells is to help us better understand the delicate mechanism of cell growth and elongation. The 40 identified phosphoproteins fall into 12 functional classes Figure 5 -A , supporting the notion that phosphorylation regulates diverse cellular processes. Among the 12 classes, protein folding and assembly, cytoskeleton and transport classes contained the higher ratio of phosphorprotein, suggesting these proteins might need more delicate regulation during the fiber elongation process.

    The A Functional classification and distribution of the 40 identified phosphorylated differentially expressed proteins. The number represents the number of protein spots in each functional catalog of the 40 phosphorylated DEPs and the DEPs not identified as phosphorylated. B and C Close-up analysis of the relationship between phosphorylation modification and the possible isoforms detected by 2-DE.

    The identified phosphoproteins are shown in red. In our previous research, we reported that many biochemical pathways were significantly regulated during the cotton fiber elongation process [18] , but the detailed molecular mechanisms of these regulations remained unknown. Based on our phosphopeptide identification results, we could now further extend our understanding of these processes to a molecular regulation level. Enolase ENO , enzyme involved in glycolysis, which catalyzes the transformation of 2-phosphoglycerate to phosphoenolpyruvate, was identified as phosphorylated Table 1.

    This finding is in full agreement with known reports that ENO is a phosphoprotein [31] , indicating that glycolysis is regulated at this step through phosphorylation during cotton fiber elongation process. Transketolase TK , a key enzyme linking the glycolysis and pentose phosphate pathways [32] was identified as a phosphoprotein.

    These results indicate that carbohydrate metabolism pathways, which play diverse and important roles in the cotton fiber elongation process [34] , may be regulated by reverse phosphorylation of important enzymes. Combined with the superior separation ability of 2-DE and the accuracy of MS identification, the different isoforms of same proteins can be mapped on a 2-DE gel [12] , [35].

    Proteomics of Biological Systems Protein Phosphorylation Using Mass Spectrometry Techniques

    Identification of the post-translation modification differences among these isoforms could then explain how they formed. This information usually cannot be easily obtained using the shotgun proteomics method but is a straightforward result of our phosphorylation site identification method. As reported previously, there are a total of 40 proteins having 2 or more isoforms detected in the 2-DE gels of elongating cotton fiber total protein [12] , [18] , of which 15 were found to have phosphorylated isoforms in this study Figures 5 -B and C.

    Among the 15 proteins, 11 have only two isoforms, and 7 of those, including UDP-L-rhamnose synthase UER , have one isoform phosphorylated and another unphosphorylated isoform. Two phosphorylated isoforms were found for 5-methyltetrahydropteroyltriglutamate—homocysteine methyltransferase MetE , and three phosphorylated isoforms were found for enolase ENO Table 1.

    These results further support the finding that reverse phosphorylation of proteins is widespread and dynamic during the cotton fiber elongation process Figure S1 and Table 1. Cotton fiber elongation is a dynamic process accompanied by major protein changes at different elongation stages.