Abstract
Proteomics is gradually complementing large shotgun qualitative studies with hypothesis-driven quantitative experiments. Targeted analyses performed on triple quadrupole instruments in selected reaction monitoring mode are characterized by a high degree of selectivity and low limit of detection; however, the concurrent analysis of multiple analytes occurs at the expense of sensitivity because of reduced dwell time and/or selectivity due to limitation to a few transitions. A new data acquisition paradigm is presented in which selected reaction monitoring is performed in two ways to simultaneously quantify and confirm the identity of the targeted peptides. A first set of primary transitions is continuously monitored during a predetermined elution time window to precisely quantify each peptide. In addition, a set of six to eight transitions is acquired in a data-dependent event, triggered when all the primary transitions exceed a preset threshold. These additional transitions are used to generate composite tandem mass spectra to formally confirm the identity of the targeted peptides. This technique was applied to analyze the tryptic digest of a yeast lysate to demonstrate the performance of the technique. We showed a limit of detection down to tens of attomoles injected and a throughput exceeding 6000 transitions in one 60-min experiment. The technique was integrated into a linear work flow, including experimental design, data acquisition, and data evaluation, enabling large scale proteomic studies.
Highlights
Proteomics is gradually complementing qualitative studies focused on protein identification relying on shotgun strategies [1, 2] with large scale quantitative experiments
The drawback of this approach is that only a limited number of compounds can be practically analyzed in one HPLC-MS run because the instrument is continuously monitoring 8 –10 transitions for each peptide no matter whether the peptide is detected from the sample or not, resulting in the waste of a significant portion of the instrument time. To overcome this issue and increase the throughput, we propose that the instrument constantly monitor only a small subset of SRM transitions for each peptide for the actual quantification and in addition confirm the peak identity using the full set of fragment ions, which are acquired in a data-dependent mode. To provide this instrument capability, we developed an innovative instrument control software, called intelligent selected reaction monitoring, that can use the specificity of a small subset of SRM transitions to quantify and intelligently trigger the full list for confirmation of target peptides, thereby allowing the simultaneous qualitative and quantitative analysis of up to 1000 peptides in a single LC-MS experiment
We describe the concept of intelligent selected reaction monitoring (iSRM) and the work flow associated to it, and we demonstrate the increased throughput facilitating the development of SRM assays and its ability to perform large scale screens targeting a respectable number of proteins
Summary
A whole yeast cell lysate was solvated in 0.1 M ammonium bicarbonate buffer containing 8 M urea at a final concentration of 1 mg/ml. For the selected reaction monitoring experiments using the iSRM instrument control software (part of the TSQ driver 2.2.0), two primary and an additional six secondary fragment ions were used for each targeted peptide. The instrument acquisition parameters, including elution time window, collision energy, and intensity threshold for triggering the data-dependent SRM scan, were generated by using Pinpoint 1.0 software (Thermo Scientific). A mixture of 10 isotopically labeled peptides (ELASGLSFPVGFK*, GAIAAAHYIR*, GILFVGSGVSGGEEGAR*, ELGQSGVDTYLQTK*, LTILEELR*, AIPGEYITYALSGYVR*, TTFINTLFQTVLK*, LVEDPQVIAPFLGK*, GISNEGQNASIK*, and LGNDDEVILFR* with the asterisk indicating 13C isotopically labeled amino acids) was run on the TSQ Vantage where we monitored all y-ions for those peptides Using these transitions, we identified the elution time for each peptide. Additional instrument parameters used for the iSRM experiments include cycle time, scan time, and dynamic exclusion set up for triggering the data-dependent SRM scan. Peak areas of all primary ions for quantification and calculated coefficients of variation (CVs) (in percent) for the repeated experiments
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call