MS3-IDQ: Utilizing MS3 spectra beyond quantification yields increased coverage of the phosphoproteome in isobaric tag experiments

摘要

Protein phosphorylation is critically important for many cellular processes, including progression through the cell cycle, cellular metabolism and differentiation. Isobaric labeling, e.g., Tandem Mass Tags (TMT), in phosphoproteomics workflows enables both relative and absolute quantitation of these phosphorylation events. Traditional TMT workflows identify peptides using fragment ions at the MS2 level and quantify reporter ions at the MS3 level. However, in addition to the TMT reporter ions, MS3 spectra also include fragment ions which can be used to identify peptides. Here, we describe using MS3 spectra for both phosphopeptide identification and quantification, a process which we term MS3-IDQ. To maximize quantified phosphopeptides, we optimize several instrument parameters, including the modality of mass analyzer (i.e., ion trap or Orbitrap), MS2 automatic gain control (AGC), and MS3 normalized collision energy (NCE) to achieve the best balance of identified and quantified peptides. Our optimized MS3-IDQ method included the following parameters for the MS3 scan: NCE=37.5 and AGC target = 1.5E5, scan range =100–2000. Data from the MS3 scan were complementary to that of the MS2 scan and the combination of these scans can increase phosphoproteome coverage by over 50%, thereby yielding a greater number of quantified and accurately localized phosphopeptides.