Apex peptide elution chain selection: a new strategy for selecting precursors in 2D-LC-MALDI-TOF/TOF experiments on complex biological samples.
نویسندگان
چکیده
LC-MALDI provides an often overlooked opportunity to exploit the separation between LC-MS and MS/MS stages of a 2D-LC-MS-based proteomics experiment, that is, by making a smarter selection for precursor fragmentation. Apex Peptide Elution Chain Selection (APECS) is a simple and powerful method for intensity-based peptide selection in a complex sample separated by 2D-LC, using a MALDI-TOF/TOF instrument. It removes the peptide redundancy present in the adjacent first-dimension (typically strong cation exchange, SCX) fractions by constructing peptide elution profiles that link the precursor ions of the same peptide across SCX fractions. Subsequently, the precursor ion most likely to fragment successfully in a given profile is selected for fragmentation analysis, selecting on precursor intensity and absence of adjacent ions that may cofragment. To make the method independent of experiment-specific tolerance criteria, we introduce the concept of the branching factor, which measures the likelihood of false clustering of precursor ions based on past experiments. By validation with a complex proteome sample of Arabidopsis thaliana, APECS identified an equivalent number of peptides as a conventional data-dependent acquisition method but with a 35% smaller work load. Consequently, reduced sample depletion allowed further selection of lower signal-to-noise ratio precursor ions, leading to a larger number of identified unique peptides.
منابع مشابه
MALDI target for improved analysis of complex proteomics samples
described. The new AnchorChipTM target provides substantial improvements to the LC-MALDI analysis of complex biological samples. The MTP AnchorChip 1536 TF can accommodate fractions collected from an extended LC gradient (up to four hours) or, alternatively, from multiple shorter LC gradients spotted in sequence. Complete MALDI-TOF/TOF analysis of all the spotted fractions can be easily achieve...
متن کاملCharacterization of the N-glycosylation Pattern of Antibodies by ESI - and MALDI mass spectrometry
Analysis of the N-glycosylation pattern on antibodies is described using complementary mass spectrometric strategies based on both, top-down ESI-UHR-TOF and bottom-up LC-MALDI-TOF/TOF. Fast LC-ESI-UHR-TOF analysis, performed on the Bruker maXisTM, provides highresolution, high-mass accuracy (confident low ppm) data for both, intact antibodies and released antibody heavy chains allowing a rapid ...
متن کاملCharacterization of the N-glycosylation Pattern of Antibodies by ESI – and MALDI Mass Spectrometry
Analysis of the N-glycosylation pattern on antibodies is described using complementary mass spectrometric strategies based on both, top-down ESI-UHR-TOF and bottom-up LC-MALDI-TOF/TOF. Fast LC-ESI-UHR-TOF analysis, performed on the Bruker maXisTM, provides highresolution, high-mass accuracy (confident low ppm) data for both, intact antibodies and released antibody heavy chains allowing a rapid ...
متن کاملIdentification of mammalian cell lines using MALDI-TOF and LC-ESI-MS/MS mass spectrometry.
Direct mass spectrometric analysis of complex biological samples is becoming an increasingly useful technique in the field of proteomics. Matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) is a rapid and sensitive analytical tool well suited for obtaining molecular weights of peptides and proteins from complex samples. Here, a fast and simple approach to cellular protein p...
متن کاملA Novel Strategy for the Analysis of Phosphopeptides, Coupling N-terminal Peptide Derivatization and Hplc Separation with Mass Spectrometry
• Here we present an N-terminal derivatization approach, using TMPP-Ac-Osu that improves the HPLC performance for small hydrophilic phosphopeptides. • This approach is compatible with LC/MS and LC/MALDI. • The chromatographic enhancement was evaluated by on-line nanoscale HPLC coupled with Electrospray on a Q-Tof PremierTM. • LC-MALDI/MS/MS analysis was also investigated. Both MS/MS on a Q-Tof ...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- Journal of proteome research
دوره 9 11 شماره
صفحات -
تاریخ انتشار 2010