Rev Anal Chem 31 (2012): 29–41 © 2012 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/revac-2011-0025 Enrichment strategies for phosphoproteomics: state-of-the-art Barbora Salovska 1,2, *, Ales Tichy 1,2 , Martina MIP molecularly imprinted polymer Rezacova 1 , Jirina Vavrova 2 and Eva Novotna 2 MOAC metal oxide affi nity chromatography MS mass spectrometry 1 Department of Medical Biochemistry , Faculty of NTA nitriloacetic acid Medicine in Hradec Kralove, Charles University in PTMs post-translational modifi cations Prague, Hradec Kr á lov é , Czech Republic , pS phosphoserine e-mail: [email protected] pT phosphothreonine 2 Department of Radiobiology , Faculty of Military Health pY phosphotyrosine Sciences, University of Defence, Hradec Kralove , RPLC reversed phase liquid chromatography Czech Republic SAX strong anion-exchange chromatography SCX strong cation-exchange chromatography * Corresponding author Introduction Abstract Protein phosphorylation is a key regulator in many biological The human genome involves approximately 30,000 protein- processes, such as homeostasis, cellular signaling and com- coding genes; the human proteome contains several million munication, transcriptional and translational regulation, and different protein effectors. This is due to alternative splic- apoptosis. The defects in this tightly controlled reversible post- ing of genes and post-translational modifi cations (PTMs). translational modifi cation have been described to contribute Several hundred PTMs are currently known, among them to genesis and progression of various diseases, emphasizing protein phosphorylation is the most studied and one of the the importance of a systematic research of this phenomenon. most important in nature (Pinkse and Heck 2010 ). Although considerable effort has been devoted to improving Protein phosphorylation is a transient, reversible PTM, which the analysis of phosphorylation by mass spectrometry, which is involved in many cellular processes including homeostasis, is currently the method of choice to study protein phospho- cellular signaling and communication, proliferation, differen- rylation, the detection and identifi cation of phosphorylation tiation, metabolism, transcriptional and translational regula- sites remains challenging because of the low abundance and tion, degradation of proteins and cell survival (Cohen 2002 ). It low ionization effi cacy of phosphoproteins in comparison with is one of the most widespread regulatory mechanisms; it has nonphosphorylated proteins. To overcome this obstacle, differ- been estimated that more than 50 % of the proteins in mam- ent enrichment strategies for phosphorylated peptides/proteins malian cells are phosphorylated at some point during their have been established and optimized for subsequent mass spec- life time (Reinders and Sickman 2005 ). Four types of phos- trometry analysis. In this review, we will give an overview of phorylation are known: O-phosphorylation which occurs the methods currently available for the enrichment of phospho- on serine, threonine and tyrosine residues, N- (Knezevic rylated proteins and peptides including immunoprecipitation, et al. 2000 ), S- (Weigt et al. 1995 ) and acyl- (Sanders et al. 1989 ) chemical derivatization and affi nity enrichment techniques. phosphorylation which are far less common and occur on histi- dine, lysine, cysteine, aspartic and glutamic residues. In eukary- Keywords: metal oxide affi nity chromatography; otic cells phosphorylation on serine, threonine and tyrosine phosphopeptide enrichment; phosphoproteomics. residues is considered to be predominant (Sickmann and Meyer 2001 ). Phosphorylation also occurs on histidine residues; a total of 6 % of the total phosphorylation in eukaryotes consists Abbreviations of phosphohistidine residues (Matthews 1995 ). Nevertheless, phosphohistidine residues are not normally observed in pro- BSA bovine serum albumin teins due to rapid hydrolysis of the phosphoryl group under DHB 2,5-dihydroxybenzoic acid acidic conditions (Hultquist 1968 ). The distribution proportions ERLIC electrostatic repulsion hydrophilic interaction of phosphoserine (pS), phosphothreonine (pT) and phospho- chromatography tyrosine (pY) sites were reported for the fi rst time in 1980 by HAP hydroxyapatite chromatography HILIC hydrophilic interaction chromatography Hunter and Sefton who determined them in chicken cells as IDA imidoacetic acid 92.19 % , 7.77 % and 0.03 % , respectively (Hunter and Sefton IMAC immobilized metal affi nity chromatography 1980 ). Since then, several studies have been performed, e.g., a LC liquid chromatography study on HeLa cells, which showed that the distribution propor- LPD liquid phase deposition tions of pS, pT and phosphotyrosine pY sites were 86.4 % , 11.8 % 30 B. Salovska et al.: Enrichment strategies for phosphoproteomics and 1.8 % (Olsen et al. 2006 ) or a study performed on the same phosphorylation have a limited dynamic range. Hence, major cell line reported by Chen et al. (2011b) , with 84.98 % , 14.26 % phosphorylation sites might be identifi ed easily, whereas minor and 0.76 % , respectively. Additionally, in Arabidopsis thaliana , sites might be diffi cult to detect. The complications caused by a popular model organism in plant biology and genetics, relative low ionization effi ciency and low abundance of phosphory- abundances of pS, pT and pY were estimated to be 85 % , 10.7 % lated proteins and peptides can be reduced by phosphospecifi c and 4.3 % (Sugiyama et al. 2008 ). This indicates that there are enrichment prior to their characterization by MS. The separation substantial differences in distribution proportions mainly due to of phosphorylated peptides from nonphosphorylated can be per- various methodical approaches and dynamic range of particular formed either on the protein level or on the peptide level. phosphoproteome used in a given study. Cellular protein phos- Tyrosine phosphorylated proteins can be enriched with the phorylation events are site-specifi c; they often occur at multiple immunoprecipitation with phosphotyrosine specifi c antibod- sites within a protein and it has been established that more than ies that have been employed successfully in some studies 100,000 phosphorylation sites may exist in the human proteome (Blagoev et al. 2004 , Rush et al. 2005 , Schumacher et al. 2007 ). (Zhang et al. 2002 ). Some of them are always quantitatively Although antibodies against specifi c phosphorylated motifs in phosphorylated, whereas others are only transiently phosphory- phosphothreonine and phosphoserine have been used in some lated up to 0.5 % (Reinders and Sickman 2005 ). studies (Gr ø nborg et al. 2002 , Zhang et al. 2002 ), immunopre- Phosphorylation is mediated by protein kinases, which com- cipitation is not capable for enriching of phosphoserine- and pose one of the largest enzyme superfamilies in higher eukary- phosphothreonine-containing proteins. Phosphoproteins can otes (Manning et al. 2002 ). It has been estimated that 2 – 3 % also be precipitated using lanthanum ions (Pink et al. 2011 , of all eukaryotic genes are coding protein kinases (Manning et Verma et al. 2011 ). Protein kinases can be specifi cally cap- al. 2002 ). The reverse reaction, dephosphorylation, is mediated tured with immobilized low-molecular weight inhibitors, e.g., by protein phosphatases. Tight cooperation of protein kinases bisindolylmaleimide compounds used by Brehmer and his col- and protein phosphatases is essential for regulation of biologi- leagues (2004). Commercial kits for the enrichment of phos- cal processes in a cell and dysregulation of these processes has phoproteins, e.g., Phosphoprotein Purifi cation Kit (QIAGEN, been described to contribute to genesis and progression of can- Hilden, Germany; Holland et al. 2011 ) or Thermo Scientifi c cer and other diseases (Blume -Jensen and Hunter 2001 ). The Pierce Phosphoprotein Enrichment Kit (Thermo Fischer dysregulation is mostly induced by mutations in genes coding Scientifi c, Rockford, IL, USA; Nilsson et al. 2010 ) are also protein kinases, overexpression of kinases or defects in negative available and used for phosphoprotein enrichment. regulatory mechanisms. Technological advances in the recent Many methods for the enrichment of phosphopeptides have past led to the development of phosphoproteomic approaches been developed so far. However, any one of them is not able to that allow researchers to identify aberrantly activated signaling yield comprehensive information about the phosphoproteome pathways and determine the appropriate therapeutic targets that of complex biological samples. Therefore, the approaches can be, for example, specifi cally targeted by small molecule described thereinafter are often combined to obtain complete kinase inhibitors (reviewed in Harsha and Pandey 2010 ). information about the phosphopeptide pool in cells, biofl uids, Mass spectrometry (MS) is currently the method of choice etc. Phosphopeptide enrichment techniques comprise chemical to describe dynamic changes in protein phosphorylation. derivatizations, affi nity enrichment methods and some alter- We covered in detail various MS approaches of phosphop- native methods, e.g., barium (Ruse et al. 2008 ) and calcium roteomic analysis in our previous work (Tichy et al. 2011 ). (Zhang et al. 2007 ) precipitation. An interesting approach Nevertheless, the MS analysis of phosphoproteins is still for identifi cation of protein kinases substrates was presented complicated because of the relatively low abundance of phos- in 2009 when phosphospecifi