Phosphorylation Analysis by Mass Spectrometry

1 Research Phosphorylation Analysis by mass Spectrometry MYTHS, FACTS, AND THE CONSEQUENCES FOR QUALITATIVE AND QUANTITATIVE MEASUREMENTS*. Hanno Steen , Judith A. Jebanathirajah **, John Rush , Nicolas Morrice , and Marc W. Kirschner . The mass spectrometric Analysis of protein phosphoryla- ever, this number is in stark contrast to the actual number of tion is still far from being routine, and the outcomes Phosphorylation sites found so far. For instance, the Phos- thereof are often unsatisfying. Apart from the inherent database ( ) currently lists 1703. problem of substoichiometric Phosphorylation , three ar- experimentally verified Phosphorylation sites for 556 different guments as to why Phosphorylation Analysis is so prob- proteins derived from eukaryotes, the human protein refer- lematic are often quoted, including (a) increased hydro- ence database ( ) lists 3652 reported phospho- philicity of the phosphopeptide with a concomitant loss rylation sites on 1240 human proteins, and PhosphoSite (www.)

2 During the loading onto reversed-phase columns, (b) se- lective suppression of the ionization of phosphopeptides ) lists 6084 non-redundant Phosphorylation in the presence of unmodified peptides, and (c) lower sites on 2430 human and mouse proteins. However, a recent ionization/detection efficiencies of phosphopeptides as study by Beausoleil et al. (1) gave some idea about the ex- compared with their unmodified cognates. Here we pres- pected number of Phosphorylation in the cell when the au- ent the results of a study investigating the validity of these thors performed a large scale phosphoproteomics study on three arguments when using electrospray ionization mass HeLa cell nuclei.

3 Although their method was biased against Spectrometry . We utilized a set of synthetic peptide/phos- basic phosphopeptides where His, Lys-Pro, and/or Arg-Pro phopeptide pairs that were quantitated by amino acid residues are in close proximity to the phosphoamino acid Analysis . Under the applied conditions none of the exper- residue, more than 2000 Phosphorylation sites on 967 nuclear iments performed supports the notions of (a) generally proteins were found. Considering the fact that protein phos- increased risks of losing phosphopeptides during the loading onto the reversed-phase column because of phorylation Analysis is of major interest in numerous labora- decreased retention and (b) the selective ionization sup- tories around the world it is surprising that more information pression of phosphopeptides.

4 The issue of ionization/ about protein Phosphorylation sites has not been gathered detection efficiencies of phosphopeptides versus their since the discovery of protein Phosphorylation . unphosphorylated cognates proved to be less straight- This raises the question as to why it is still such a challenge forward when using electrospray ionization because to perform unbiased protein Phosphorylation Analysis . One no evidence for decreased ionization/detection efficien- inherent reason is the generally low Phosphorylation stoichi- cies for phosphopeptides could be found.

5 Molecular & ometry of most of the proteins such that phosphopeptides are Cellular Proteomics 5:172 181, 2006. grossly underrepresented in the generated complex peptide mixtures (see below). In addition, numerous reasons can be listed for not being able to identify and localize a phosphoryl- Protein Phosphorylation is one of the most prevalent intra- ation site in a given protein, which affects all stages of phos- cellular protein modifications that is of pivotal importance in phorylation Analysis , sample preparation, Analysis , and numerous cellular processes including cell differentiation, data interpretation.

6 Examples for problems during the sample proliferation, and migration. It is estimated that 30% of all preparation include omission of phosphatase inhibitor, proteins in a cell are phosphorylated at any given time. How- overestimation of the degree of Phosphorylation because of the lack of information about the Phosphorylation stoichiom- From the Department of Pathology, Harvard Medical School and etry when using phosphospecific antibodies, and/or underes- Children's Hospital, Boston, Massachusetts 02115, Department of timation of the heterogeneity of the Phosphorylation due to Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, Neurobiology Program, Children's Hospital, Boston, Massa- nonspecific 32P-labeling.

7 Chusetts 02115, **MDS Sciex, Concord, Ontario L4K 4V8, Canada, However, even if it was ensured that a particular protein Cell Signaling Technology, Beverly, Massachusetts 01915, and sample is phosphorylated by using other methods such as MRC Protein Phosphorylation Unit, University of Dundee, radiolabeling it is often the case that the mass spectrometric Dundee, United Kingdom DD1 5EH. Received, May 12, 2005, and in revised form, September 30, 2005. Analysis does not provide any information about the sites of Published, MCP Papers in Press, October 4, 2005, DOI Phosphorylation .

8 Three main reasons are commonly used to explain as to why the phosphopeptides were missed during 172 Molecular & Cellular Proteomics 2006 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at Myths and Facts about Phosphopeptide Analysis by MS. the mass spectrometric Analysis : (i) increased hydrophilicity chromatograms (XICs)1 of RNYSVGS and RNYpSVGS (Table and hence reduced retention of phosphopeptides on re- I, Peptide species 2), the shortest peptide/phosphopeptide versed-phase materials, (ii) selective suppression of their ion- pair investigated.

9 Although there is a considerable overlap in ization/detection efficiencies in the presence of large amounts the elution profiles, the phosphopeptide clearly elutes after of unphosphorylated peptides, and (iii) lower detection effi- the unphosphorylated cognate. Of the three doubly phospho- ciencies of phosphopeptides as compared with their unphos- rylated peptides in this test set, two phosphopeptides eluted phorylated cognates. In this study we tried to scrutinize these significantly later than the singly phosphorylated or unphos- different arguments often brought forward with the aim to phorylated peptide from the reversed-phase column; one ex- draw the attention to the most prevalent problem(s) of mass ample is shown in Fig.

10 1B. The third doubly phosphorylated spectrometric Phosphorylation Analysis and to improve the peptide with the sequence DQAVpTEpYVATR (Table I, Pep- Analysis where it is most fruitful/promising. tide species 27) was the only peptide whose modified form eluted before its unmodified complement (Fig. 1C). It was MATERIALS AND METHODS noted that this particular peptide was the only phosphopep- All solvents used were of HPLC-grade quality from Burdick and tide in our sample set in which the number of phospho moi- Jackson (VWR International). All chemicals were purchased from eties exceeded the number of basic amino acid residues.