MS3-IDQ: Utilizing MS3 Spectra beyond Quantification Yields Increased Coverage of the Phosphoproteome in Isobaric Tag Experiments

Abstract

Protein phosphorylation is critically important for many cellular processes, including progression through the cell cycle, cellular metabolism, and differentiation. Isobaric labeling, for example, 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 that can be used to identify peptides. Here we describe using MS3 spectra for both phosphopeptide identification and quantification, a process that 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 = 1.5 × 10⁵, and scan range = 100-2000. Data from the MS3 scan were complementary to those of the MS2 scan, and the combination of these scans can increase phosphoproteome coverage by >50%, thereby yielding a greater number of quantified and accurately localized phosphopeptides.

Keywords: MS3 sequencing; TMT; isobaric labeling; phosphoproteomics.

Figure 1. MS3-IDQ Phosphopeptide Workflow

To generate our phosphopeptide sample, intact mouse brain was solubilized in SDS lysis buffer. Following reduction, alkylation and digestion, phosphopeptides were enriched using IMAC and labeled with TMTzero. Fusion Lumos MS settings were adjusted to allow for optimal phosphopeptide identification and localization at both the MS2 and MS3 stage.

Figure 2. Optimizing MS3-NCE for MS3-IDQ

We varied the HCD Normalized Collision Energy (NCE) to determine the optimal settings to use in MS3 spectra-based quantification. Error bars represent ± standard deviation (n=2).

Figure 3. Complementarity of MS2 and MS3 scans

Example of a phosphopeptide (TVFAGAVPVLPAS#PPPK, 2+) for which A) MS2 and B) MS3 scans each detect unique fragment ions, which would increase the quality of the identification. C) Table summarizing characteristics of both scans.

Figure 4. Fractionated sample shows that MS3-IDQ provides additional unique, quantified, and localized phosphopeptides

A) In a fractionated study, we note that the MS3 scan provided more identifications than the MS2 scan. B) An unfractionated TMT-labeled phosphopeptide sample was analyzed in two separate laboratories and showed increase in quantified phosphopeptides with lower NCE. Normalized collision energy settings of 37.5 and 55 for MS3 fragmentation were compared between laboratories. Measurements were performed in triplicate.