Optimization of Higher-Energy Collisional Dissociation Fragmentation Energy for Intact Protein-Level Tandem Mass Tag Labeling

Abstract

Isobaric chemical tag labeling (e.g., TMT) is a commonly used approach in quantitative proteomics, and quantification is enabled through detection of low-mass reporter ions generated after MS2 fragmentation. Recently, we have introduced and optimized an intact protein-level TMT labeling platform that demonstrated >90% labeling efficiency in complex samples with top-down proteomics. Higher-energy collisional dissociation (HCD) is commonly utilized for isobaric tag-labeled peptide fragmentation because it produces accurate reporter ion intensities and avoids loss of low mass ions. HCD energies have been optimized for isobaric tag labeled-peptides but have not been systematically evaluated for isobaric tag-labeled intact proteins. In this study, we report a systematic evaluation of normalized HCD fragmentation energies (NCEs) on TMT-labeled HeLa cell lysate using top-down proteomics. Our results suggested that reporter ions often result in higher ion intensities at higher NCEs. Optimal fragmentation of intact proteins for identification, however, required relatively lower NCE. We further demonstrated that a stepped NCE scheme with energies from 30% to 50% resulted in optimal quantification and identification of TMT-labeled HeLa proteins. These parameters resulted in an average reporter ion intensity of ∼4E4 and average proteoform spectrum matches (PrSMs) of >1000 per RPLC-MS/MS run with a 1% false discovery rate (FDR) cutoff.

Pilot Evaluation of the Long-Term Reproducibility of Capillary Zone Electrophoresis–Tandem Mass Spectrometry for Top-Down Proteomics of a Complex Proteome Sample

“…If a large-scale study is needed, for example, comparing multiple samples with more than 20 CZE-MS runs, then the Phase II condition should be considered. Alternatively, stable isotopic labeling techniques (e.g., tandem mass tags 46 ) can be employed. In this case, we may not need to worry about the Phase I or Phase II condition because the relative quantification is performed based on the data within the same CZE-MS runs.…”

Very Low-Pressure CID Experiments: High Energy Transfer and Fragmentation Pattern at the Single Collision Regime

“…The results highlight that CID under conventional conditions is the result of a high number of collisions, even as many as 20–50 collision events. In proteomics, it has become fashionable to use ‘composite spectra’, which are a combination of low- and high-energy CID spectra [ 18 , 20 , 21 , 47 ]. The result is qualitatively similar to the low-pressure CID spectra studied here.…”

“…Increasing the collision energy increases the internal energy of the colliding ions, and that in turn increases the degree of fragmentation, changes the ratio of various fragment ion abundances, and may also change fragmentation routes. Varying and optimizing the collision energy has various practical applications, such as determination of binding energies [ 8 , 9 ], differentiation of isomers [ 10 , 11 , 12 ], characterization of oligonucleotides [ 13 ] and small molecules [ 14 , 15 , 16 , 17 ], and various proteomic applications [ 18 , 19 , 20 ] including quantitation [ 21 ].…”

Second Special Issue on Methods for Omics Research: Proteome Research and Beyond

“…Here, Guo et al follow up on TMT-labeling at the protein level by evaluating HCD fragmentation settings to optimize the quantitation of intact proteoforms through reporter ion intensities. 18…”

“…Quantitation of intact proteoforms is usually performed in a label-free manner, but recent work from the Wu lab, , among others, has taken advantage of isobaric tandem mass tags (TMT), which are routinely used for bottom-up approaches, to label intact proteins. Here, Guo et al follow up on TMT-labeling at the protein level by evaluating HCD fragmentation settings to optimize the quantitation of intact proteoforms through reporter ion intensities …”