David Horn

Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry

Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique that provides a more comprehensive coverage of peptide sequences and posttranslational modifications. Here, we evaluated the use of ETD for a global phosphoproteome analysis. In all, we identified a total of 1,435 phosphorylation sites from human embryonic kidney 293T cells, of which 1,141 (≈80%) were not previously described. A detailed comparison of ETD and collision-induced dissociation (CID) modes showed that ETD identified 60% more phosphopeptides than CID, with an average of 40% more fragment ions that facilitated localization of phosphorylation sites. Although our data indicate that ETD is superior to CID for phosphorylation analysis, the two methods can be effectively combined in alternating ETD and CID modes for a more comprehensive analysis. Combining ETD and CID, from this single study, we were able to identify 80% of the known phosphorylation sites in >1,000 phosphorylated peptides analyzed. A hierarchical clustering of the identified phosphorylation sites allowed us to discover 15 phosphorylation motifs that have not been reported previously. Overall, ETD is an excellent method for localization of phosphorylation sites and should be an integral component of any strategy for comprehensive phosphorylation analysis.

Guidelines for reporting the use of mass spectrometry in proteomics

Joeri Borstlap1, Glyn Stacey2, Andreas Kurtz3, Anja Elstner3, Alexander Damaschun1, Begoña Arán4 & Anna Veiga4,5 1CellNet Initiative, Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité– Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. 2The UK Stem Cell Bank, National Institute for Biological Standards and Control, Blanch Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK. 3Cell Therapy Group, BerlinBrandenburg Center for Regenerative Therapies (BCRT), Charité–Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. 4Banc de Linies Cellulars, Centre de Medicina Regenerativa de Barcelona (CMRB), C/Dr. Aiguader 88, 08003-Barcelona, Spain. 5Institut Universitari Dexeus, Passeig de la Bonanova 67, 08017-Barcelona, Spain. e-mail: joeri.borstlap@b-crt.de

Guidelines for reporting the use of mass spectrometry informatics in proteomics.

volume 26 number 8 august 2008 nature biotechnology are addressed in the MIAPE-MS (mass spectrometry) module, the latest version of which can be obtained from the MIAPE home page. Note also that these guidelines do not cover all the available features of a protein and peptide identification and characterization tool (e.g., some of the less frequently used parameters, types of spectra or other experimental data); subsequent versions may have expanded coverage, as will almost certainly be the case for all MIAPE modules. These guidelines will evolve in step with progress in research. The most recent version of MIAPE-MSI is available at http://www.psidev.info/miape/msi/ and the content is replicated here as supplementary information (Supplementary Guidelines and Supplementary Table 1). To contribute or to track the process to remain ‘MIAPE compliant’, browse the website at http:// www.psidev.info/miape/.

Recommendations for Mass Spectrometry Data Quality Metrics for Open Access Data (Corollary to the Amsterdam Principles)

Policies supporting the rapid and open sharing of proteomic data are being implemented by the leading journals in the field. The proteomics community is taking steps to ensure that data are made publicly accessible and are of high quality, a challenging task that requires the development and deployment of methods for measuring and documenting data quality metrics. On September 18, 2010, the United States National Cancer Institute convened the “International Workshop on Proteomic Data Quality Metrics” in Sydney, Australia, to identify and address issues facing the development and use of such methods for open access proteomics data. The stakeholders at the workshop enumerated the key principles underlying a framework for data quality assessment in mass spectrometry data that will meet the needs of the research community, journals, funding agencies, and data repositories. Attendees discussed and agreed up on two primary needs for the wide use of quality metrics: 1) an evolving list of comprehensive quality metrics and 2) standards accompanied by software analytics. Attendees stressed the importance of increased education and training programs to promote reliable protocols in proteomics. This workshop report explores the historic precedents, key discussions, and necessary next steps to enhance the quality of open access data. By agreement, this article is published simultaneously in the Journal of Proteome Research, Molecular and Cellular Proteomics, Proteomics, and Proteomics Clinical Applications as a public service to the research community. The peer review process was a coordinated effort conducted by a panel of referees selected by the journals.