2012 ASMS Fall Workshop: Mass Spectrometry-Based Phosphorylation Analysis and Phosphoproteomics
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- Tao, W.A. & Coon, J. J. Am. Soc. Mass Spectrom. (2013) 24: 464. doi:10.1007/s13361-012-0562-0
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The 2012 ASMS Fall Workshop was held on November 8–9 at the Hyatt Harborside Hotel in Boston, MA. This year, the topic focused on mass spectrometry-based phosphorylation analysis and phosphoproteomics. The workshop was organized and chaired by Professors W. Andy Tao, from Purdue University and Joshua Coon, from University of Wisconsin-Madison, and brought together scientists from around the world; there were 10 experts in the field who gave keynote lectures, and ~150 scientists from all aspects of the field (university faculty, graduate students, post-docs, industry researchers, etc.), who attended as interested participants. The workshop covered basic instrumentation and strategies for targeted and global protein phosphorylation analysis. The speakers often used real biological applications to demonstrate the four major topics addressed: (1) sample preparation; (2) data acquisition, identification, and quantitation; (3) software tools for phosphoproteomics, and (4) analyses of signaling network. Each major topic was discussed in a three-part lecture, with three separate speakers (each spoke for 45 min), followed by a 30-min panel discussion. The panel discussion periods, which were led by the three instructors who gave the lecture on the topic, allowed for participants to engage with experts and attendees alike. When asked, many attendees said that this style of lecture/discussion was an exceptional workshop format.
Mass spectrometry has become the major force in phosphorylation analyses today. With advances in high resolution mass spectrometry and bioinformatics, identification of phosphorylation sites in high throughput has become relatively routine work in many university facilities. The protocols and working procedures for MS, however, are far from standard. The same protocol that works fine with one biological sample and one researcher, may lead to much poorer results with a different sample, in a different lab, or even just with a different researcher working on the same sample in the same lab. This inconsistency was demonstrated by Coon at the beginning of the workshop; he presented data, generated by different graduate students in his lab, which showed a wide range of different phosphopeptide identifications made while using the same sample, the same protocol, and the same instrument. With such inconsistent results in academic phosphoproteomics, a majority of industry R&D teams are still observing from the sideline. A discussion in the ASMS education committee in 2010 concluded that a workshop on this specific topic would greatly benefit the scientific community as a whole.
The workshop opened on Thursday morning with an introductory overview of the current status of phosphoproteomics, given by Dr. Ruedi Aebersold (ETH, Switzerland). His talk encompassed the evolution of phosphorylation, tools for phosphorylation site analysis, and the discrepancy between current large phosphoproteomic identifications and functional validation. Dr. Aebersold extracted six fundamental topics related to phosphoproteomics: identification of phosphoproteins and phosphorylation sites, stoichiometry of phosphosites and kinase-substrate relationships, and biochemical and cellular function of phosphorylation events. Some of these, namely identifications, are relatively mature, while others, such as the stoichiometry of interactions, remain the subject of intense study; the last of these six, biochemical and cellular functions of phosphorylation events, appear to be the farthest out in terms of functional study.
Dr. Martin Larsen (University of Southern Denmark) followed this insightful introduction with a lecture on sample preparation, focusing on current approaches to phosphopeptide enrichment. Enrichment is essential in order to efficiently isolate phosphopeptides, which is one of the most critical steps in mass spectrometry-based phosphorylation analysis. Larsen’s lecture centered on TiO2-based strategies, and examined different protocols for optimal selectivity. Dr. Jarrod Marto (Dana-Farber Cancer Institute, Boston, MA) then discussed LC methods, which fractionate phosphopeptides, offering an insightful evaluation on when and how best to employ these methods.
The Thursday afternoon session was dedicated to phosphopeptide data acquisition, identification, and quantitation. Dr. Joshua Coon (University of Wisconsin, Madison, WI) presented on the fundamentals of phosphopeptide fragmentation, discussing a variety of common dissociation techniques on current commercial instrumentation. Dr. Scott Gerber (Dartmouth College, NH) followed this with a lecture on the identification of phosphorylation sites in shotgun proteomic experiments. He discussed the impact of scoring, FDR estimation, and high mass accuracy on phosphopeptide identification, based primarily on the Sequest algorithm. Dr. Jesper Olsen (University of Copenhagen, Denmark) then reviewed quantitative approaches to protein phosphorylation measurements, including stable isotopic labeling and label-free quantitation. As one of the major experts contributing to stable isotope labeling with amino acids in cell culture (SILAC), Olsen reported on some recent developments and new applications of SILAC, including Super SILAC for clinical samples.
Dr. Olsen also started off the Friday morning session, in which he spoke on software tools in phosphoproteomics. This lecture delved into the computerized algorithms used to extract raw data from MS analysis, localize phosphorylation sites, and determine kinase motifs. He used Graphical Proteomics data eXplorer (GroX) as an example of a software-user interface for data annotation in phosphoproteomic projects.
Dr. Forest White (MIT, Boston, MA) covered the pervasive study of protein tyrosine phosphorylation, a signaling network of pathways. Using EGFR signaling as a model, White linked quantitative phosphorylation changes to cellular functions, such as cell growth and cell viability. To overcome relatively poor reproducibility of data-dependent acquisition, Dr. White offered independent data acquisition (IDA), and multi-reaction monitoring (MRM) as potential solutions. Dr. Steve Gygi (Harvard Medical School, Boston, MA) then focused on how to measure relative and absolute phosphorylation stoichiometries. The measurement of actual phosphorylation changes is complicated by proteins’ own expression changes, and, based on his lab’s data, he showed that more than 25 % of protein phosphorylation changes are due to changes in protein expression. Dr. Gygi subsequently introduced an innovative proteomic approach to measure absolute stoichiometries: half of a yeast sample was treated with a phosphatase, and then changes in non-phosphopeptide abundance were detected.
The final session of the workshop showcased several phosphoproteomic projects attempting to dissect signaling networks. Dr. Philippe Roux (University of Montreal, Canada) reported on his group’s studies with ribosomal S6 kinase (RSK)-dependent signaling pathways. The RSK family contains Ser/Thr kinases, known downstream signaling molecules in the mitogen-activated protein kinase (MAPK) pathway, but whose substrates in cancer cell growth and proliferation are not known. Combining SILAC-based quantitative phosphoproteomics with RSK inhibitors and RNA interference (RNAi), the Roux Group has identified a large number of phosphorylation events that are RSK-dependent. Many of them are potential RSK substrates, but remain to be characterized.
Next was W. Andy Tao (Purdue University, IN), who reviewed high throughput methods for identifying kinase substrates. Current approaches include: in vitro kinase assay; using synthetic peptides; cell extracts or phage display; interaction-based screenings, such as yeast two-hybrid and affinity purification; and mass spectrometry-based screenings. Dr. Tao introduced an integrated proteomic approach, termed kinase assay linked phosphoproteomics (KALIP), for identifying direct kinase substrates. This strategy increases selectivity and specificity by enriching formerly phosphorylated peptides and proteins derived directly from a cellular lysate for in vitro kinase reaction. The Tao Group applied the KALIP method to spleen tyrosine kinase (SYK), as a model, and identified a number of SYK direct substrates with high confidence.
Dr. Ruedi Aebersold wrapped up the conference with a discussion of his focus: dissecting yeast and human kinase-substrate networks based on in vitro kinase reactions and targeted mass spectrometry (i.e., MRM). His group’s work demonstrated that quantitative phosphoproteomics, based on integrated mass spectrometric methods, can establish the network link between genotype and phenotype.
This workshop provided a unique opportunity for attendees and lecturers to interact and exchange ideas. The wide selection of topics, and presence of the experts in the field, was essential to its success, and this effective consolidation of information was lauded by all participants. According to the post-workshop survey, the workshop met or exceeded attendees’ expectations. This was the first time, in the history of the ASMS fall workshop that attendees had such positive feedback and would look forward to attending another workshop on a different topic.