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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 Copyright © 2009, Bioinfo Publications, International Journal of Pharmaceuticals Analysis, ISSN: 0975-3079, Volume 1, Issue 2, 2009 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
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