TMTpro Complementary Ion Quantification Increases Plexing and Sensitivity for Accurate Multiplexed Proteomics at the MS2 Level

Abstract

Multiplexed proteomics is a powerful tool to assay cell states in health and disease, but accurate quantification of relative protein changes is impaired by interference from co-isolated peptides. Interference can be reduced by using MS3-based quantification, but this reduces sensitivity and requires specialized instrumentation. An alternative approach is quantification by complementary ions, the balancer grouppeptide conjugates, which allows accurate and precise multiplexed quantification at the MS2 level and is compatible with most proteomics instruments. However, complementary ions of the popular TMT-tag form inefficiently and multiplexing is limited to five channels. Here, we evaluate and optimize complementary ion quantification for the recently released TMTpro-tag, which increases complementary ion plexing capacity to eight channels (TMTproC). Furthermore, the beneficial fragmentation properties of TMTpro increase sensitivity for TMTproC, resulting in ∼65% more proteins quantified compared to TMTpro-MS3 and ∼18% more when compared to real-time-search TMTpro-MS3 (RTS-SPS-MS3). TMTproC quantification is more accurate than TMTpro-MS2 and even superior to RTS-SPS-MS3. We provide the software for quantifying TMTproC data as an executable that is compatible with the MaxQuant analysis pipeline. Thus, TMTproC advances multiplexed proteomics data quality and widens access to accurate multiplexed proteomics beyond laboratories with MS3-capable instrumentation.

Super-resolution mass spectrometry enables rapid, accurate, and highly-multiplexed proteomics at the MS2-level

“…This is exciting, as most multiplexed proteomics experiments are currently performed with 50 000 resolution settings, and the additional 20 ms transient time seems negligible. We therefore expect SR experiments with higher plexing capability to have similar speed and sensitivity as the 1 Da TMTproC analysis 5, 12, 22 . Typically, the CVs further improve, when the quantitative information form peptides are integrated onto the protein level.…”

“…An alternative approach to tackle peptide co-isolation and the associated reporter ion ratio-compression problem is MS2-level complement reporter ion quantification. 11, 12, 13 Following this method, the species to be monitored are the complementary reporter ions – the remainders of the precursor ions labeled with the normalization parts and the reactive groups. For TMTpro/TMT the complementary ion clusters will have one charge less than the isolated precursor ions and thus will be detected in an even higher mass range, up to 2 000 m/z , and, sometimes, beyond that.…”

Differential nuclear import sets the timing of protein access to the embryonic genome

“…2), we could enrich the nuclei at various embryonic stages and determine the nuclear fraction of each protein (FN) by quantifying its signal in the supernatant and the flow-through via multiplexed proteomics (Fig. 2a) 29,30 . We determined when half of the protein had entered the embryonic nuclei using a sigmoidal fit of the protein's FN over time (Tembryo1/2).…”

A Causal Model of Ion Interference Enables Assessment and Correction of Ratio Compression in Multiplex Proteomics

“…Wühr et al described an alternative quantification strategy in which the complementary fragment ion cluster in the MS2 spectrum is used for the quantitative profiling 13 . However, most current MS instruments lack the required resolution to separate heavy N and C isotope mass difference in the higher m/z region of the complementary ions which reduces the multiplexing capabilities to a maximum of 8 channels 14 . Another hurdle lies in the complementary ion formation itself, as currently available isobaric labeling systems are not optimized to generate complementary fragment ions 15 Finally, there are strategies that address ratio compression in silico.…”