Proteomics/Post-translational Modification/Phosphoproteomics

Chapter Edited and Updated by: Richard Rodrigues E-mail: rrr5868@rit.edu

This Section:

Introduction
Phosphoproteomics is the branch of proteomics that deals with identification and characterization of proteins with an attached phosphate group. Protein phosphorylation is a reversible post-transcriptional modification which plays an important role in signal transduction, protein function and localization. The phosphorylation of serine, threonine and tyrosine residues is the most common in mammalian cells, whereas the phopshorylation of histidine and aspartate is rare in eukaryotes.

Detection and characterization
The detection and characterization of phosphoproteomics has been traditionally done with kinases, phosphatases, biochemical and genetic studies. More recently, antibodies, 2D gel electrophoresis and mass spectrometry are being widely used because of their ability to give specific results with high throughput. Due to the usage of mass spectrometry in phosphoprotemics analysis, there has been tremendous developments in the areas of sample preparation, instrumentation, quantitative methodology, and informatics.

Applications
The phosphoproteomic analysis of cells and tissues provide insights about cell signaling, cell differentiation status , new phosphorylation motifs , kinase-substrate specificity and number and types of protein phosphorylations. Studies on cancer phosphoproteomics might provide us with prospectice biomarkers.

Phosphoproteomics for the masses
Reviewer: Richard Rodrigues

Main Focus

 * The main focus is to give an overview about Phosphoproteomic analysis using Mass Spectrometry, current applications and the future challenges for the field.

New Terms

 * Shotgun Proteomics: method which involves digestion of protein mixture, peptide separation by liquid chromatography and tandem mass spectrometry to identify proteins in the mixture. (http://en.wikipedia.org/wiki/Shotgun_proteomics)
 * Collisional Dissociation: method which involves collision of an accelerated molecular ion with an inert gas molecule resulting in smaller fragments of the charged ion. (http://en.wikipedia.org/wiki/Collision-induced_dissociation)
 * Electron-based dissociation: method which involves capture or transfer of an electron to a charged ion to induce its fragmentation. (1: http://en.wikipedia.org/wiki/Electron_transfer_dissociation 2: http://en.wikipedia.org/wiki/Electron_capture_dissociation)
 * False discovery rate: it is a ratio of number of false positives to the sum of number of false positives and number of true positives. (http://en.wikipedia.org/wiki/False_discovery_rate)
 * Phosphopeptide enrichment: is the isolation and concentration of peptides with phosphate groups attached for further analysis via mass spectrometry. (source: http://en.wikipedia.org/wiki/Phosphoproteomics)

Summary

 * [[Image: L-Phosphoserine.png|thumb| L-Phosphoserine ]]	[[Image: Affinity-column-2010-09-04.png|thumb| Affinity column-chromatography ]] Protein phosphorylation plays an important role in signal transduction across species, with kinases and phosphatases accounting for 2–4% of eukaryotic proteomes. Application of the Mass Spectrometry (MS) technologies have allowed for phosphoprotein analysis in a wide variety of biological contexts, thus overcoming the limitations of traditional biochemical techniques. Uses of MS technology in different studies have revealed insight into the cells of whole organisms, the role of phosphorylation events in determining the fate of individual cells, the role of individual protein phosphorylation events on signaling networks within the cell, and identification of the phosphorylation events on protein substrates of various kinases.


 * Phosphopeptide enrichment strategies like affinity-based approaches (immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and strong cation exchange (SCX), anti-pTyr immunoaffinity methods) and other alternate approaches (BEMA (ß-elimination/Michael addition), phosphoramidate chemistry (PAC), phosphate metal affinity chromatography (PMAC) and calcium phosphate precipitation) are currently being used inline with MS to prevent suppression of unstable phosphopeptides during ionization. The tandem MS approach involves separation of a phosphopeptide mixture by capillary liquid chromatography followed by electrospray ionization (ESI) that generates multiple protonated gas-phase peptide cations. This process of precursor selection, dissociation, and fragment ion mass analysis is iterated on analyte species as they elute from the liquid chromatography column. This MS based approach helps to identify the peptide primary sequence and position of the phosphoryl modifications.


 * Peptide fragmentation can be achieved by collision-activated dissociation (CAD), electron capture dissociation (ECD) and electron transfer dissociation (ETD). Quantitation methods like metabolic labeling, isobaric tagging, isotope tagging and isotope dilution are the widely used to produce peptides with slight differences in mass by incorporation of heavy stable isotopes. Recently, label-free approaches have also been utilized for phosphopeptide quantitation. The intensity of the mass spectral peaks is then used to determine the relative amount of the peptide in one condition versus the other. The conventional shotgun proteomics strategies applicable for phosphoproteomics involves the use of target–decoy database searching at both the peptide and protein level making possible the control of the false discovery rate (FDR) of a dataset.


 * Various software tools are available to identify site localization (e.g. Ascore, PhosphoScore, Phosphinator and SLomo), data sharing/mining (e.g. PhosphoSitePlus, Phospho.ELM, Phosphorylation Site Database (PHOSIDA) and PhosphoPep). High-resolving power mass analysis can be used to distinguish isobaric modifications like phosphorylation and sulfonation. Ongoing developments in MS technology and related instrumentation will not only increase the large sets of phosphoproteomic data, but also increase the dynamic range and sensitivity. This will allow the detection with lower amount of samples and identification of lower-abundance phosphorylation events. Using informatics to utilize the available data and get biological insights will help in the study of phosphoproteomics.

Relevance to a Traditional Proteomics Course

 * The paper relates to the course of proteomics as it gives the overview of mass spectroscopy in phosphoproteomic analysis.