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

摘要

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 proteinsfrom a HeLa cell extract. Using a standard data-dependent approach, we identified some specificpeptides from this extract which were also commercially available in their AQUA form (use forabsolute quantitation). For some of the peptides, we observed a non-linear response between theintensity and the added quantity which was then fitted using a quadratic fit. All AQUA peptidesspiked into a mix of 3 μg of the HeLa cell digest extract were detected down to 16 fmol. We placed anemphasis on peptide detection which, in this study, is performed using a combination of propertiessuch as three specific Q3-like ion signatures (for a given Q1-like selection) and co-elution with theAQUA peptide counterparts. Detecting a peptide without necessarily identifying it using a searchengine imposes less constraint in terms of tandem mass (MS/MS) spectra purity. An example isshown where a peptide is detected using those criteria but could not be identified by Mascot due to itslower abundance. To complement this observation, we used a cross-correlation analysis approach inorder 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. Anin silico analysis of the human trypsinosome allows the evaluation of how unique are the sets offeatures that we are using for peptide detection.