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.
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, which allows accurate and precise multiplexed quantification at the MS2 level and is compatible with the most widely distributed 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 plexing capacity to eight channels (TMTProC). We find that the beneficial fragmentation properties of TMTPro increase quantification signal five-fold compared to TMT. This increased sensitivity results in ~65% more proteins quantified compared to TMTPro-MS3 and even slightly outperforms TMTPro-MS2. Furthermore, TMTProC quantification is more accurate than TMTPro-MS2 and even superior to TMTPro-MS3. To demonstrate the power of TMTProC, we analyzed a human and yeast interference sample and were able to quantify 13,290 proteins in 24 fractions. Thus, TMTProC advances multiplexed proteomics data quality and widens access to accurate multiplexed proteomics beyond laboratories with MS3-capable instrumentation.
In tandem mass spectrometry (MS2)-based multiplexed quantitative proteomics, the complement reporter ion approaches (TMTc and TMTproC) were developed to eliminate the ratio-compression problem of conventional MS2level approaches. Resolving all high m/z complement reporter ions (∼6.32 mDaspaced) requires mass resolution and scan speeds above the performance levels of Orbitrap TM instruments. Therefore, complement reporter ion quantification with TMT/TMTpro reagents is currently limited to 5 out of 11 (TMT) or 9 out of 18 (TMTpro) channels (∼1 Da spaced). We first demonstrate that a Fusion TM Lumos TM Orbitrap can resolve 6.32 mDa-spaced complement reporter ions with standard acquisition modes extended with 3 s transients. We then implemented a super-resolution mass spectrometry approach using the least-squares fitting (LSF) method for processing Orbitrap transients to achieve shotgun proteomics-compatible scan rates. The LSF performance resolves the 6.32 mDa doublets for all TMTproC channels in the standard mass range with transients as short as ∼108 ms (Orbitrap resolution setting of 50,000 at m/z 200). However, we observe a slight decrease in measurement precision compared to 1 Da spacing with the 108 ms transients. With 256 ms transients (resolution of 120,000 at m/z 200), coefficients of variation are essentially indistinguishable from 1 Da samples. We thus demonstrate the feasibility of highly multiplexed, accurate, and precise shotgun proteomics at the MS2 level.
Protein turnover is a critical regulatory mechanism for proteostasis. However, proteome-wide turnover quantification is technically challenging and, even in the well-studied E. coli model, reliable measurements remain scarce. Here, we quantify the degradation of ~3.2k E. coli proteins under 12 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. Furthermore, we show that protein degradation rates are generally independent of cell division rates, and we used knockout experiments to assign substrates to the known ATP-dependent proteases. Surprisingly, we find that none are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Thus, we introduce broadly applicable technology for protein turnover measurements. We provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to decipher the control of proteostasis.
The complementary reporter ion approaches overcome the ratio-compression problem of multiplexed proteomics and enhance the accuracy of protein quantification at the MS2 level. However, resolving the high m/z complementary ions encoded via mass defects in carbon and nitrogen (∼6.32 mDa mass difference in TMT/TMTpro) requires mass resolution and scan speeds above the performance levels offered by even the state-of-the-art Orbitrap™ instruments. Therefore, complement ion quantification (TMTc/TMTproC) is currently limited to only 5 (TMT) or 9 (TMTpro) channels (∼1 Da spaced) versus 11 or 18, respectively, when analyzing the conventional low m/z reporter ions. Here, we first demonstrate the feasibility of the highly-plexed complementary ion quantification in the high m/z range by enabling ultra-high-resolution (UHR) capabilities on a commercial Orbitrap mass spectrometer. The UHR performance required 3 s time-domain transients, which were acquired and processed with the high-performance data acquisition-processing system FTMS Booster X2 externally interfaced with the Orbitrap Fusion™ Lumos™ instrument. Despite validating the TMTc approach for the whole mass range, the UHR capability is incompatible with the practical data acquisition times (scan speeds) for liquid chromatography (LC)-based proteomics. We thus implemented a super-resolution mass spectrometry approach based on the least-squares fitting (LSF) that resolves even the 6.32 mDa doublets for all TMTproC channels in the whole mass range with time-domain transients as short as ∼108 ms (resolution setting of 50 000 at m/z 200). A more demanding, quantitatively accurate TMTproC performance is provided at the resolution setting of 120 000 at m/z 200 (256 ms time-domain transients). This advance enables accurate, LC timescale-compatible, and highly-multiplexed proteomics at the MS2 level.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.