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Quantitative analysis of low‐abundance peptides in HeLa cell cytoplasm by targeted liquid chromatography/mass spectrometry and stable isotope dilution: emphasising the distinction between peptide detection and peptide identification
Author(s) -
Le Bihan Thierry,
Grima Ramon,
Martin Sarah,
Forster Thorsten,
Le Bihan Yann
Publication year - 2010
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.4487
Subject(s) - chemistry , peptide , mass spectrometry , chromatography , tandem mass spectrometry , hela , elution , isotope dilution , quadrupole ion trap , ion trap , biochemistry , cell
We present the application of a targeted liquid chromatography/mass spectrometry (LC/MS) approach developed on a linear ion trap for the evaluation of the abundance of cytoplasmic proteins from a HeLa cell extract. Using a standard data‐dependent approach, we identified some specific peptides from this extract which were also commercially available in their AQUA form (use for absolute quantitation). For some of the peptides, we observed a non‐linear response between the intensity and the added quantity which was then fitted using a quadratic fit. All AQUA peptides spiked into a mix of 3 µg of the HeLa cell digest extract were detected down to 16 fmol. We placed an emphasis on peptide detection which, in this study, is performed using a combination of properties such as three specific Q3‐like ion signatures (for a given Q1‐like selection) and co‐elution with the AQUA peptide counterparts. Detecting a peptide without necessarily identifying it using a search engine imposes less constraint in terms of tandem mass (MS/MS) spectra purity. An example is shown where a peptide is detected using those criteria but could not be identified by Mascot due to its lower abundance. To complement this observation, we used a cross‐correlation analysis approach in order to separate two populations of MS/MS fragments based on differences in their elution patterns. Such an approach opens the door to new strategies to analyse lower intensity peptide fragments. An in silico analysis of the human trypsinosome allows the evaluation of how unique are the sets of features that we are using for peptide detection. Copyright © 2010 John Wiley & Sons, Ltd.