Molecular & Cellular Proteomics Blog

Robert Chalkley

The ability to reliably assign sites of post-translational modification is one of the more challenging aspects of mass-spectrometry-based proteomic analysis.  Higher quality data is generally required to identify a site of modification than is required to identify the peptide and its modification state, such that it is commonly the case that a modification can be reliably assigned but the exact location of the modification within the peptide may be ambiguous.

Data analysis is performed by one of a range of database search engines, but these are usually optimized for peptide identification rather than modification site assignment.  For example, software normally apply an intensity threshold to the peak list to reduce the number of noise peaks, leading to a higher percentage of peaks being matched; e.g. it is statistically more significant if 25 out of the 30 most intense peaks match than 30 out of the 60 most intense peaks.  However, those five extra peak matches may be important for isolating the modification site.  The second weakness of most search engines for modification site assignment is that they do not clearly indicate which modification site assignments are reliable and which are ambiguous; i.e. where the data is consistent with the site reported, but another site is equally well supported.  Hence, further analysis of spectra identifying PTM sites is generally required.  This is currently most commonly performed by ‘manual verification’, which can be reliable if the person is experienced in spectral analysis, but is a subjective process and is going to be inconsistent.  Alternatively, a second piece of software specifically designed for modification site assignment may be employed.  This software is not going to be as powerful and sophisticated as an expert manual reviewer, but will provide more consistent results and it may be possible to assign a measure of reliability to the site assignments.

Recognizing this weakness, the MCP journal publication guidelines for studies reporting PTM site assignments require authors to make available annotated spectra for all biological PTM site assignments, so that readers can view the spectra and decide whether they agree with the assignment. This raises the questions: how often do people disagree with other people’s interpretations and how reliable are PTM site assignments reported in the literature?

The ‘Proteome Informatics Research Group’ (iPRG) of ABRF is seeking to answer these questions in their 2010 study.  They are distributing a phosphopeptide dataset and are asking participants to report modification site assignments and methods they used to derive these sites.  Results will be published at the ABRF conference in March 2010, will be presented at other conferences and will be made available through the ABRF website.  As there are a number of phosphorylation publications in the literature reporting hundreds or even thousands of phosphorylation sites, these results will be very interesting for assessing the reliability of already published sites.

Participation in this study is not restricted only to members of ABRF, so anyone interested in partaking should send an e-mail to iPRG2010@gmail.com.  Data will be distributed mid-November and results should be returned by Monday January 10^th 2010.

David E. Gloriam, Sandra Orchard, Daniela Bertinetti, Erik Bjorling, Erik Bongcam-Rudloff, Julie Bourbeillon, Andrew R. Bradbury, Antoine de Daruvar, Stefan Dubel, Roanld Frank, Toby J. Gibson, Niall Haslam, Friedrich W. Herberg, Tara Hiltke, Jorg D. Hoheisel, Samuel Kerrien, Manfred Koegl, Zoltan Konthur, Bernhard Korn, Ulf Landegren, Silvere van der Maarel, Luisa Montecchi-Palazzi, Sandrine Palcy, Henry Rodriguez, Sonja Schweinsberg, Volker Sievert, Oda Stoevesandt, Michael J. Taussig, Mathias Uhlen, and Christer Wingren

Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology and diagnostics, as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome-scale applications. This situation has triggered several initiatives involving large-scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific sub-proteomes are being pursued by members of HUPO (Plasma and Liver Proteome Projects) and the U.S. National Cancer Institute (cancer associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one online warehouse and find all available affinity reagents from different providers together with documentation that facilitate easy comparison of their cost and quality. However, in contrast to for example nucleotide databases among which data are synchronized between the major data providers; current PAR producers, quality control centres and commercial companies all use incompatible formats hindering data exchange. Here we propose PSI-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the HUPO Proteomics Standards Initiative (PSI) and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-MI, which is a widely accepted and established community standard for molecular interactions data. Further information and documentation is available on the PSI-PAR website.

See full report  in press (free) at MCP online.

Comments welcome.

N. Leigh Anderson, Norman G. Anderson, Terry W. Pearson, Christoph H. Borchers, Amanda G. Paulovich, Scott D. Patterson, Michael Gillette, Ruedi Aebersold, and Steven A. Carr

The lack of sensitive, specific, multiplexable assays for most human proteins is the major barrier impeding development of candidate biomarkers into clinically useful tests. Recent progress in mass spectrometry-based assays for proteotypic peptides, particularly those with specific affinity peptide enrichment, offers a systematic and economical path to comprehensive quantitative coverage of the human proteome. A compete suite of assays, e.g., two peptides from the protein product of each of the ~20,500 human genes (here termed the hPDQ project), would enable rapid and systematic verification of candidate biomarkers, and lay a quantitative foundation for subsequent efforts to define the larger universe of splice variants, post-translational modifications, protein-protein interactions and tissue localization.

Read full paper in press at MCP Online

Comments welcome.

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