Phosphoproteomics: Recent Advances in Analytical Techniques

International Journal of Pharmaceuticals Analysis, ISSN: 0975-3079, Volume 1, Issue 2, 2009, pp-31-36

Phosphoproteomics: Recent advances in analytical techniques

Gomase V.S. and Ramu Akella Department of Biotechnology, Padmashree Dr. D.Y. Patil University, Navi Mumbai, 400614, India

Abstract - Post-genomic biology seeks identification and quantification of multiple proteins from complex mixtures and the research is still on. Despite recent progress in high-throughput proteomics, proteomic analysis of post-translationally modified [PTM] proteins remains particularly challenging. Several strategies for isolating phosphoproteins are explored herein. Quantification of phosphoproteins seems to be a novel solution to identify the underlying disease mechanisms, mostly cancer. Keywords - phosphorylation, enrichment, chemical modification, SILAC, mass spectrometry, HILIC

Introduction Techniques Phosphoproteomics is a branch of proteomics Of late, there is an increasing interest in that focuses on deriving a comprehensive view of phosphoproteomics as reflected by a the extent & dynamics of protein phosphorylation considerable number of works describing various by way of identifying & characterizing proteins strategies, including the use of different isotopic that contain a phosphate group as a post- technologies [e.g., isotope-coded affinity tag, translational modification. The addition of stable isotope labeling by amino acids in cell phosphate is a key reversible modification culture], protein separation techniques [e.g., gel- catalyzed by protein kinases, and is known as based versus liquid chromatography-based ‘phosphorylation’. Protein kinases account for methods], and chemical modifications [e.g., β- 1.7% of the human genome and 40% of all the elimination and esterification] to identify proteins may be assumed to be phosphorylated phosphoproteins and/or phosphorylation sites in at any given time. Interestingly, the first evidence a global fashion [21, 32]. Conventional of Ser/Thr/Tyr protein phosphorylation in a phosphoproteomic analysis used radioactive member of the “third domain of life” was reported labeling, followed by two-dimensional gel in the extreme halophilic archaeon Halobacterium electrophoresis, phosphopeptide mapping by salinarum in 1980, using 32 P radiolabeling. This thin-layer chromatography and electrophoresis, was followed by discovering the existence of or sequencing by Edman degradation [30, 34]. In protein phosphorylation in the extreme due course, they laid more emphasis on site- acidothermophile Sulfolobus acidocaldarius [1, directed mutagenesis, mass spectrometry and 16, 28, 39]. Traditional biochemical and genetic enriching phosphoproteins. Emergent analyses of phosphoproteins, and of the kinases technologies like microarrays and fluorescence- and phosphatases that modify them, have based single-cell analysis are characterized by provided a barrage of information about signaling high sensitivity and high throughput, but require pathways. Since these methods focus on one prior knowledge of particular phosphoprotein protein at a time, they are not readily amenable targets. Mass spectrometry-based methods offer to understand the complexity of protein rapid, selective and sensitive analysis and phosphorylation or how individual discovery of new phosphoproteins. phosphoproteins function in the context of signaling networks. Recent advancements in Enriching Phosphoproteins & analytical technology, particularly mass Phosphopeptides spectrometry, coupled with availability of Since many phosphoproteins [notably signaling biological databases have fuelled the active study intermediates] are low-abundance proteins of many phosphoproteins and phosphorylation phosphorylated at sub-stoichiometric levels, sites at once [28]. In sharp contrast to several enrichment strategies have been conventional expression profiling studies, developed in consonance with mass phosphoproteomics potentially provides two spectrometry [33, 45]. The commonly used additional layers of information. First, it provides enrichment techniques are immobilized metal clues on what protein or pathway might be affinity chromatography [IMAC], phosphoprotein activated [because a change in the isotope-coded affinity tag [PhIAT] and phosphorylation status of proteins almost always phosphoprotein isotope-coded solid-phase tag reflects a change in protein activity]. Second, it [PhIST]. IMAC has undergone incremental indicates what proteins might be potential drug improvements like the replacement of traditional targets [because phosphoproteomics focuses on metal chelating resins with cellulose powder, proteins that have kinase activities or are silica monolithic supports or poly [glycidyl substrates of kinases] [21]. methacrylate/divinylbenzene] [GMD] derivatized with imino-diacetic acid and bound Fe [III] as a material [4]. The use of aluminium hydroxide or addition of 40% 1, 1, 1, 3, 3, 3- hexafluoroisopropanol to buffers have improved

Phosphoproteomics: Recent advances in analytical techniques

the overall efficiency of IMAC [38]. Nowadays, The technology has also been successfully phosphopeptide enrichment is a highly applied to the study of epidermal growth factor accelerated process, based on the availability of [EGF] and fibroblast growth factor [FGF] several commercial kits like PhosphoProtein signaling. SILAC is advantageous because it Purification Kit [Qiagen], Pro-Q Diamond removes false positives, reveals large-scale Phosphoprotein enrichment kit [Invitrogen / kinetics of proteomes and uncovers important Molecular Probes] and BD Phosphoprotein points along signaling pathways directly [34, 38]. enrichment kit [BD Biosciences] [10, 13, 15, 28]. In a typical SILAC experiment, cells representing Sequential elution from IMAC [SIMAC] is also two different biological conditions are grown in being used to sequentially separate media supplemented with ‘light’ or ‘heavy’ monophosphorylated and multiphosphorylated isotope-containing amino acids. Metabolic peptides from complex biological samples [46]. incorporation of labeled amino acids into all Current techniques for peptide enrichment are proteins from cells of one population, and mainly based on various flavours of metal affinity subsequent combination of labeled and chromatography. Non-specific binding by acidic unlabeled samples in equal ratios, enables proteins and peptides, and favoured enrichment quantification of proteins from the two samples of poly-phosphorylated species tend to plague based on the intensities of the light and heavy this system. Chemical modification is considered peptides. Within the same mass spectrometric necessary to bypass this deficiency [43]. experiment, tandem mass spectrometry [MS/MS] can be carried out to obtain sequence information Chemical Modification for protein identification. Thus, targeted Chemical modification of phosphorylated proteomic comparisons can be achieved in a residues has so far proven the most successful high-throughput way. With evolution, SILAC has phosphoproteomic strategy, and entails two emerged as a very powerful method to study cell approaches. In the first approach, base-mediated signaling, protein-protein interactions and β-elimination of phosphate from phospho-Ser or - regulation of gene expression [17, 24, 48]. Thr residues permits subsequent addition of biotinylated probes at pre-phosphorylated sites. Mass Spectrometry Though not applicable to tyrosine Any mass spectrometer has three quintessential phosphorylation, this method has seen some parts: an ionization source, a mass analyzer and success for the global analysis of phosphorylated a detector. Electron spray ionization [ESI], matrix- proteins from Arabidopsis , but it still suffers from assisted laser desorption/ionization [MALDI] a low but significant background of β-elimination and/or surface-enhanced laser desorption / from glycosylated and free Ser residues [25, 31, ionization [SELDI] have been used for 43]. In the second reported approach, a short phosphoproteomic analysis of the adipose series of reactions based on reversible secretome. Time-of-flight [TOF] and quadrupole phosphoramidate chemistry was applied to TOF [QTOF] instruments, as also ion traps and introduce a thiol group at phosphorylated sites for linear quadrupole ion trap-Fourier transforms using them as a handle to introduce secondary [LTQ-FTs] have been constructively used. labels. This method has recently been elaborated Sustained efforts are on to improve four aspects for the identification of phosphorylated proteins in of mass spectrometry-based phosphoproteomic Drosophila melanogaster Kc167 cells and for use analyses, namely improved accuracy and speed, in general chemical phosphoproteomics. In improved separation of complex mixtures, addition to these two methods, some progress computational refinement of the signals obtained, has resulted in the analysis of phosphorylated & integrating the flood of results in intelligent proteins tagged metabolically with a γ- databases [9, 29, 40]. Protein characterization by thiophosphate ATP analogue [6, 7, 37, 43]. mass spectrometry is usually done by the “bottom-up” or “top-down” approach. However, Stable Isotope Labeling by Amino Acids in characterization of phosphoproteins uses the Cell Culture [SILAC] “bottom-up” approach, wherein protease Separation of identical biochemical species by digestion is followed by qualitative and/or mixing cellular lysates derived from isotopically quantitative analysis by mass spectrometry [MS]. distinct media sources forms the basis of Electron capture dissociation [ECD] and electron metabolic labeling. SILAC is a mass transfer dissociation [ETD] are instrumental in spectrometry-based, metabolic labeling fragmenting the peptide backbone leaving the technique that paved way for the identification phosphoserine/phosphothreonine intact. They and quantification of complex protein mixtures. It have also been used constructively in “top-down” differs from other metabolic labeling strategies experiments to sequence phosphoproteins. ETD since only a specific amino acid is isotopically identifies 60% more phosphopeptides than labeled. It has been purportedly effective for collision-induced dissociation [CID]. However, a characterization of cellular signaling and protein- combination of ETD & CID can effectively be protein interactions of receptor tyrosine kinases. used for a more comprehensive analysis. Till

Gomase VS and Ramu Akella

date, ECD has been exclusively coupled with analysis is being used to generate Fourier transform ion cyclotron resonance mass phosphorylation site-specific antibodies that spectrometry [FTICR-MS], the most expensive could be used as reagents to develop antibody MS instrumentation available [12, 27, 42]. array/chip or for immunohistochemistry on tissue Developments in MS have frequently been microarrays [TMA] in molecular pathology [5, 21]. plagued by inconsistent reproducibility arising One very recent and interesting application of from automated selection of precursor ions for these phosphorylation site–specific antibodies fragmentation, identification and quantification. was in single cell profiling of signaling networks On one hand, false positives are avoided by high using multivariable flow cytometry. The human stringency criteria based on MS/MS data, while, embryonic stem cell phosphoproteome was on the other hand, these criteria induce many recently revealed by electron transfer dissociation false negatives. A new MS-based strategy, based tandem mass spectrometry. This was done by on multiple reaction monitoring of stable isotope- IMAC enrichment followed by analysis via labeled peptides, was used to quantify temporal nanoflow reversed-phase LC-MS/MS on a phosphorylation profiles of 222 tyrosine spectrometer that had been modified to perform phosphorylated peptides following pervanadate ETD. Several phosphorylation site repositories or EGF treatment. 88% of the signaling nodes curated by researchers are popping up every were reproducibly quantified, as against 34% in now and then, thereby accelerating conventional information-dependent analysis [3, phosphopeptide identification & discovery. The 23]. PHOSIDA database contains 8969 human phosphorylated sites, gathered from collective Phosphoprotein Profiling by Pa-GeLC-MS/MS literature reports. The NetworKIN database This involved a combination of phosphoprotein integrates consensus substrate motifs with affinity chromatography, denaturing gel context modeling to improve prediction of cellular electrophoresis, tandem mass spectrometry, and kinase-substrate reactions. It can also be used to informatics analysis. Native phosphoproteins study phosphorylation-modulated interaction were selectively isolated from a G2/M phase networks at the global and the molecular level yeast whole cell extract using a phosphoprotein [22, 41]. To assist in LC-MS/MS analysis, the affinity matrix – Pro-Q Diamond resin [Molecular PhosphoPIC [PhosphoPeptide Identification & Probes – Invitrogen]. This was followed by 1-D Compilation] software has been created to PAGE separation, proteolysis and ESI LC- perform a variety of functions including MS/MS [PA-GeLC-MS/MS], and generated 131 automated selection and compilation of proteins. The Pro-Q eluate was separated into phosphopeptide identifications from multiple MS two fractions by size [less than 100 kDa & greater levels [MS n]. It has also been used for the than 100 kDa] before PAGE and ESI LC-MS/MS estimation of dataset false discovery rate [FDR], analyses. The component proteins were then and application of appropriate cross-correlation queried against databases to reveal the [XCorr] filters. CID studies have revealed that a correlation between protein-protein interactions & LTQ-Orbitrap LC-MS/MS platform can identify protein phosphorylation [15]. In another study, a approximately 3 times more phosphopeptides multidimensional chromatography technology than Q-TOF LC-MS/MS instrumentation. This combining IMAC, hydrophilic interaction may be attributed to the versatility of the Orbitrap, chromatography [HILIC] and reversed-phase LC which features high resolution [up to 150,000], was used to analyze DNA damage in the yeast high mass accuracy [2-5 ppm], a mass-to-charge Saccharomyces cerevisiae . 8764 resultant range of 6000, and a dynamic range greater than phosphopeptides were obtained from 2278 that of a low resolution instrument [3, 8, 47]. The phosphoproteins, proving sufficient in-depth use of titanium dioxide [TiO 2], zirconium dioxide phosphoproteome analysis. A recent study of [ZrO 2] and niobium pentoxide [Nb 2O5] has led to HILIC, reversed phase-high performance liquid greater selectivity in enriching phosphopeptides. chromatography [RP-HPLC] and strong cation Miniaturized technologies like capillary and exchange [SCX] chromatography revealed that microfluidic platforms have greatly sped up HILIC could ensure better orthogonal separation bioanalytical and biological applications, with for unphosphorylated peptides. As suited to added sensitivity and throughput-related phosphoproteomic analyses, HILIC columns demands [11, 18, 38]. Global quantification of prefer to retain hydrophilic molecules, thereby phosphoproteins has become much easier with reducing sample complexity [2, 26, 36]. iTRAQ & AQUA. iTRAQ is an in vitro labeling procedure and can be applied to clinical samples Current Research such as serum, urine and tumor tissues. AQUA Of late, enrichment of phosphotyrosine- provides for absolute quantification of containing proteins via monoclonal phosphoproteins, unlike the relative level of antiphosphotyrosine antibodies has been a quantification produced by SILAC or iTRAQ [12]. notable recent success. Experimental data from Another phosphoproteomic approach liquid chromatography-based mass spectrometry corresponds to the identification of several

International Journal of Pharmaceuticals Analysis, ISSN: 0975-3079, Volume 1, Issue 2, 2009 33 Phosphoproteomics: Recent advances in analytical techniques

extracellular signal-regulated kinase [ERK] available to determine the targets of a single mitogen-activated protein [MAP] kinase kinase against its homologues without resorting substrates, using a combination of steroid to pleiotropic kinase knockouts [43]. Tyrosine receptor fusion system, IMAC, 2D differential gel phosphorylation prominently features in cancer electrophoresis [2D-DIGE] & phosphomotif- biology, since they serve as attractive drug specific antibodies. The phosphorylation of targets and molecular cancer markers. As such, various nucleoporins by ERK could herald novel the tumour phosphoproteome is a rich mine of theories in the regulation of the potential biomarkers. The SILAC method has nucleocytoplasmic transport [14]. unveiled the role of the spleen tyrosine kinase Syk in phosphorylating E-cadherin, for proper Technical Challenges localization of p120-catenin at adherens Low stoichiometry has proved a major junctions. Subsequent identification of the impediment in global phosphoproteomic analyses biochemical pathways regulated by Syk in cancer [35]. A prime challenge is the cost-effective cells could offer valuable insights into tumor production of large quantities of a wide range of formation and progression. Targeting tyrosine highly purified and phosphorylation site–specific kinases like the receptors Met and EphA2, the antibodies. High-density antibody array cytoplasmic tyrosine kinase Fak, and pseudo- construction is a tough task, & requires large tyrosine kinase SgK223 may be of significant amount of protein samples for analysis. Also, a therapeutic value in human colorectal carcinoma considerable amount of technical inertia has to [CRC] [17, 19, 21, 44]. be overcome before clinically designing and applying a high-throughput antibody/protein chip. Conclusion Routine clinical application of phosphoproteomics Developments in the field of phosphoproteomics could be realistically achieved using the Aptamer have been fuelled by the need to simultaneously technology and semi-high throughput TMAs. monitor many different phosphoproteins within Currently, efforts are on to make TMAs from the signaling networks that coordinate responses frozen tissue blocks, so that protein antigens can to changes in the cellular environment. be better preserved than on paraffin blocks [21]. Phosphotyrosine-directed signaling analyses are The presence of single- or double-stranded increasingly being used to provide robust nucleic acids in IMAC beads to a loss of measurements for functional biological approximately 50% of phosphopeptides during interpretation of drug action on signaling and enrichment. Acetonitrile precipitation was a phenotypic outcomes. While phosphoproteomics preferential strategy to remove nucleic acids from will greatly expand our knowledge about the protein samples prior to IMAC [20]. numbers and types of phosphoproteins, its Phosphoprotein profiling serves well to improve greatest promise is the rapid analysis of entire the statistical significance of protein identification. phosphorylation based signaling networks. The Also, the integrity and conformational stability of enormity of the protein phosphorylation profile intact proteins makes them a feasible fodder for has created many a challenge for future polyacrylamide gel electrophoresis [PAGE] researchers. Creating a phosphoprotein-specific separations. However, phosphopeptide antibody chip may bode well for cancer studies in enrichment could suffer a setback if the time to come. phosphorylation sites are misidentified. Co- purification of associated, non-phosphorylated Abbreviations peptides is also a deviating factor [15]. post-translationally modified [PTM], immobilized metal affinity chromatography [IMAC], Applications phosphoprotein isotope-coded affinity tag Recent developments in chromatographic and [PhIAT], phosphoprotein isotope-coded solid- mass spectrometric techniques have spurred phase tag [PhIST], poly [glycidyl phosphoproteomics to move beyond mere methacrylate/divinylbenzene] [GMD], sequential experiments, to a stage where complex biological elution from IMAC [SIMAC], stable isotope issues can be resolved [35]. The next big thing labeling by amino acids in cell culture [SILAC], could be a combination of phosphoproteomics epidermal growth factor [EGF], fibroblast growth with enzyme-substrate engineering. This involves factor [FGF], Electron spray ionization [ESI], mutating the ATP-binding site of a specific kinase matrix-assisted laser desorption/ionization such that it will additionally accept a bulky ATP [MALDI], surface-enhanced laser analogue. If a chemical tag is incorporated into desorption/ionization [SELDI], time-of-flight this analogue at the gamma phosphate the [TOF], quadrupole TOF [QTOF], linear protein substrates of the kinase will be tagged at quadrupole ion trap-Fourier transforms [LTQ- the site of modification, enabling their enrichment FTs], mass spectrometry [MS], Electron capture and identification. This technology would be a dissociation [ECD], electron transfer dissociation potent tool for the analysis of kinase networks [ETD], collision-induced dissociation [CID], because there is no other de novo method Fourier transform ion cyclotron resonance mass

Gomase VS and Ramu Akella

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International Journal of Pharmaceuticals Analysis, ISSN: 0975-3079, Volume 1, Issue 2, 2009 35 Phosphoproteomics: Recent advances in analytical techniques

Table 1- Methods for enriching phosphoproteins & phosphopeptides Method Comments Chemical Modification

Phosphorylated amino acids are derivatized by β-elimination or carbodiimide Affinity Tagging to introduce tags; β-elimination strategy is limited to P-Ser and P-Thr, and also occurs with O-glycosylated residues.

Analog-specific kinases are used selectively to phosphorylate substrates in vitro or in vivo ; the phosphate analogs are then derivatized to generate a Bio-orthogonal Affinity Purification hapten for immunoprecipitation. This method requires expression and/or isolation of an engineered kinase.

Perfluoroalkyl groups are selectively coupled to P-Ser and P-Thr using β- Fluorous Affinity Tagging elimination, modified peptides are enriched with fluorous-functionalized stationary phase. This method is highly selective for derivatized peptides.

Chemical modification of P-Ser and P-Thr introduces lysine analogs and Phosphospecific Proteolysis cleavage by lysine-specific proteases. This method allows direct identification of phosphorylation site without sequencing the phosphopeptide.

Proteins are phosphorylated with ATP γS; thiophosphates are alkylated to Thiophosphorylation & Affinity form linkages with biotin or solid supports. This method requires in vitro Tagging phosphorylation and most kinases utilize ATP γS poorly.

Direct Enrichment

Anti-P-Tyr antibodies have proven very useful for the enrichment of P-Tyr- Anti-phosphotyrosine Antibodies containing proteins; they have been used alone or in combination with IMAC. They have been used to enrich P-Tyr peptides.

Anti-phosphoserine & Anti- Anti-P-Ser and anti-P-Thr antibodies have been used; but currently are less phosphothreonine Antibodies useful than anti-P-Tyr antibodies. Strong cation exchange chromatography has been used for the large-scale Cation Exchange identification of phosphorylation sites. This method selects for peptides phosphorylated on a single residue.

Immobilized Metal Affinity Introduction of an esterification step greatly enhances the selectivity of this Chromatography [IMAC] method, which is very useful, has been widely used and can be automated.