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In preparation for the ABRF’03 meeting in Denver, the Proteomics Research Group (PRG) embarked on its second study. For the ABRF-PRG’03 study, we built upon our first study sample (ABRF-PRG’02) using some of the same proteins, but in this case we were evaluating methods for locating post-translational modifications (PTMs). The objective for the participants was to identify the proteins, find the phosphopeptides, and locate the actual sites of phosphorylation within those peptides. To prepare this sample we digested PDI and BSA with trypsin prior to spiking the digests with two synthetic phosphopeptides (p1, p2). Thus, the sample sent out to participants consisted of 5 pmols PDI, 0.2 pmols BSA, 1 pmol p1 and 1 pmol p2. This study was opened to nonmembers as well as members, resulting in a more than 50% response from those requesting samples, up from the typical response of about 35%. By tabulating information such as type of equipment used, experience, and particularly techniques, we hoped to identify a method or methods that could be routinely used to successfully find phosphopeptides and identify their specific phosphorylation sites. When the data were all tabulated, we found that locating phosphopeptides and the specific sites is neither easy nor straightforward and that no single method consistently stood out as the “right” way to do it. This leaves the door wide open for further methods development in this critical area.
These results were presented at ABRF’03 as a poster and formed the basis of the oral presentation by Tom Neubert, a PRG member. In addition, we decided to invite one of the three labs that obtained the correct identifications to give a brief talk on their particular method. It was not known by the PRG who this lab was, due to the anonymous nature of the data, until the person had agreed to present and identified himself. This invitation was accepted by Kenneth Standing from the University of Manitoba in Winnipeg, Canada. A fine presentation was given of the various steps taken by their lab to identify the major protein, find the peptides, and locate the phosphorylation sites. A great deal of time and work was put into the project by his lab, which is indicative of the problems surrounding identification of phosphorylation sites. This helped to support the PRG’s conclusion that identification of PTMs is not an insignificant undertaking. Our presentations were very well-attended (standing room only in a room with capacity for 325) and considered significant enough to merit front page coverage in the February 17 issue of ProteoMonitor, a weekly paper with the latest in proteomics.
The findings for the ABRF-PRG’03 study are posted on the web site in PDF format and the related article is expected in the next issue of JBT. We sincerely thank all of those who participated in this year’s study.
As the year progresses, the Proteomics Research Group is busy planning the strategy for our next study. We plan to open the study to ABRF members and nonmembers alike, as we did for the ABRF-PRG’03 study, to again attract a large participant distribution and response. The letter introducing the new study will be sent to all members in late July or early August. Sample distribution will again be in September for those who request to participate in the study. We hope to have an even larger proportion of participants returning their data for this study and look forward to presenting the results at ABRF’04 in Portland, Oregon in February of 2004.
The current PRG members are David Arnott, Mary Ann Gawinowicz, Jeffrey Kowalak, Bill Lane (EB liaison), Thomas Neubert, Kaye Speicher, Christoph Turck, and Karen West. Jeff and Karen are new to the PRG this year, filling the positions left by Kathy Stone and Raymond Grant who stepped down after 2 years each. We wish to thank Kathy and Ray for their many contributions, and we also would like to acknowledge Len Packman for all his exceptional help and advice as our EB liaison for 2001 and 2002. Many thanks for all your hard work!