A benchmarking protocol for intact protein-level Tandem Mass Tag (TMT) labeling for quantitative top-down proteomics

Guo, Yanting; Yu, Dahang; Cupp‐Sutton, Kellye A.; Liu, Xiaowen; Wu, Si

doi:10.1016/j.mex.2022.101873

Cited by 3 publications

(1 citation statement)

References 12 publications

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“…An optimized protein-level TMT labeling protocol is reported here. , Briefly, low molecular weight (<100 kDa) HeLa cell lysate proteins were enriched using 100 kDa MWCO spin filters and concentrated using 10 kDa MWCO spin filters. Low MW HeLa proteins were denatured by urea, reduced by TCEP, alkylated by IAA, buffer-exchanged to 100 mM TEAB buffer (pH 8.5), and concentrated to >1 μg/μL using 10 kDa MWCO spin filters.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2023

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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.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2023

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Characterizing Age-related Changes in Intact Mitochondrial Proteoforms in Murine Hearts using Quantitative Top-Down Proteomics

Ramirez-Sagredo,

Sunny,

Cupp-Sutton

et al. 2024

Preprint

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Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and the prevalence of CVDs increases markedly with age. Due to the high energetic demand, the heart is highly sensitive to mitochondrial dysfunction. The complexity of the cardiac mitochondrial proteome hinders the development of effective strategies that target mitochondrial dysfunction in CVDs. Mammalian mitochondria are composed of over 1000 proteins, most of which can undergo post-translational protein modifications (PTMs). Top-down proteomics is a powerful technique for characterizing and quantifying all protein sequence variations and PTMs. However, there are still knowledge gaps in the study of age-related mitochondrial proteoform changes using this technique. In this study, we used top-down proteomics to identify intact mitochondrial proteoforms in young and old hearts and determined changes in protein abundance and PTMs in cardiac aging. METHODS: Intact mitochondria were isolated from the hearts of young (4-month-old) and old (24-25-month-old) mice. The mitochondria were lysed, and mitochondrial lysates were subjected to denaturation, reduction, and alkylation. For quantitative top-down analysis, there were 12 runs in total arising from 3 biological replicates in two conditions, with technical duplicates for each sample. The collected top-down datasets were deconvoluted and quantified, and then the proteoforms were identified. RESULTS: From a total of 12 LC-MS/MS runs, we identified 134 unique mitochondrial proteins in the different sub-mitochondrial compartments (OMM, IMS, IMM, matrix). 823 unique proteoforms in different mass ranges were identified. Compared to cardiac mitochondria of young mice, 7 proteoforms exhibited increased abundance and 13 proteoforms exhibited decreased abundance in cardiac mitochondria of old mice. Our analysis also detected PTMs of mitochondrial proteoforms, including N-terminal acetylation, lysine succinylation, lysine acetylation, oxidation, and phosphorylation. CONCLUSION: By combining mitochondrial protein enrichment using mitochondrial fractionation with quantitative top-down analysis using ultrahigh-pressure liquid chromatography (UPLC)-MS and label-free quantitation, we successfully identified and quantified intact proteoforms in the complex mitochondrial proteome. Using this approach, we detected age-related changes in abundance and PTMs of mitochondrial proteoforms in the heart.

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Toward the analysis of functional proteoforms using mass spectrometry-based stability proteomics

et al. 2023

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Functional proteomics aims to elucidate biological functions, mechanisms, and pathways of proteins and proteoforms at the molecular level to examine complex cellular systems and disease states. A series of stability proteomics methods have been developed to examine protein functionality by measuring the resistance of a protein to chemical or thermal denaturation or proteolysis. These methods can be applied to measure the thermal stability of thousands of proteins in complex biological samples such as cell lysate, intact cells, tissues, and other biological fluids to measure proteome stability. Stability proteomics methods have been popularly applied to observe stability shifts upon ligand binding for drug target identification. More recently, these methods have been applied to characterize the effect of structural changes in proteins such as those caused by post-translational modifications (PTMs) and mutations, which can affect protein structures or interactions and diversify protein functions. Here, we discussed the current application of a suite of stability proteomics methods, including thermal proteome profiling (TPP), stability of proteomics from rates of oxidation (SPROX), and limited proteolysis (LiP) methods, to observe PTM-induced structural changes on protein stability. We also discuss future perspectives highlighting the integration of top-down mass spectrometry and stability proteomics methods to characterize intact proteoform stability and understand the function of variable protein modifications.

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A benchmarking protocol for intact protein-level Tandem Mass Tag (TMT) labeling for quantitative top-down proteomics

Cited by 3 publications

References 12 publications

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

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

Characterizing Age-related Changes in Intact Mitochondrial Proteoforms in Murine Hearts using Quantitative Top-Down Proteomics

Toward the analysis of functional proteoforms using mass spectrometry-based stability proteomics

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