In-Depth Analysis of C Terminomes Based on LysC Digestion and Site-Selective Dimethylation

Du, Xiaoxian; Fang, Changming; Yang, Lijun; Bao, Huimin; Zhang, Lei; Yan, Guoquan; Lu, Haojie

doi:10.1021/acs.analchem.8b05338

Cited by 16 publications

(21 citation statements)

References 33 publications

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“…The obtained results were compared with those of three recently reported Cterminomics studies using HeLa lysates. 23,26,29 As summarized in Table 1, our method was superior to these reported methods in both sensitivity and efficiency. To deepen the analysis, we used 200 μg of HeLa peptides and isolated the protein C-terminal peptides by Ce-MOLEX followed by high-pH reversed-phase LC fractionation.…”

Section: Hela C-terminome Analysismentioning

confidence: 81%

“…The Supporting Information is available free of charge on the ACS Publications website http://pubs.acs.org This study Du et al 23 Li et al 26 Wang et al 29 Sample amount for isolation…”

Section: Supporting Informationmentioning

confidence: 99%

“…14 To date, several methods have been developed to isolate protein C-terminal peptides from digested protein mixtures. [15][16][17][18][19][20][21][22][23][24][25][26][27][28] These methods can be roughly divided into two types, involving positive and negative selection. Positive selection targets protein C-terminal carboxy groups for labeling with oxazolone or carboxypeptidase 16,17 , although the labeling efficiency of these methods is incomplete or biased by the nature of the amino acid residues at the protein C-termini.…”

Section: Introductionmentioning

confidence: 99%

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One-step Isolation of Protein C-terminal Peptides from V8 Protease-digested Proteins by Metal Oxide-based Ligand Exchange Chromatography

Nishida

Ishihama

2021

Preprint

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We have developed a one-step method to isolate　protein C-terminal peptides from V8 protease-digested　proteins by metal oxide-based ligand-exchange (MOLEX)　chromatography. V8 protease cleaves the C-terminal　side of Asp and Glu, affording a digested peptide with　two carboxy groups at the C-terminus, whereas the　protein C-terminal peptide has only one α-carboxy　group. In MOLEX chromatography, a stable chelate is　formed between dicarboxylates and metal atoms, so that　the non-terminal (i.e., internal) peptide is retained,　whereas the protein C-terminal peptide flows through the MOLEX column. After optimization of the MOLEX chromatographic conditions, 1619 protein C-termini were identified from 30 μg of peptides (10 μg each, in triplicate) derived from human HeLa cells by means of nanoLC/MS/MS. When the MOLEX-isolated sample from 200 μg of HeLa peptides was further divided into six fractions by high-pH reversed-phase LC prior to nanoLC/MS/MS, 2202 protein C-termini were identified with less than 3% contamination with internal peptides. We believe this is the largest coverage with the highest purity reported to date in human protein C-terminomics. This fast, simple, sensitive and selective method to isolate protein C-terminal peptides should be useful for profiling protein C-termini on a proteome-wide scale.

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Section: Hela C-terminome Analysismentioning

confidence: 81%

“…The Supporting Information is available free of charge on the ACS Publications website http://pubs.acs.org This study Du et al 23 Li et al 26 Wang et al 29 Sample amount for isolation…”

Section: Supporting Informationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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One-step Isolation of Protein C-terminal Peptides from V8 Protease-digested Proteins by Metal Oxide-based Ligand Exchange Chromatography

Nishida

Ishihama

2021

Preprint

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“…Another effort we've been made is to improve the C‐TAILS strategy based on Lys C digestion and site‐selective dimethylation. [ 68 ] As shown in Figure 5, Lys C specifically hydrolyzed peptide bonds C‐terminal to the lysine residues. After Lys C digestion, internal peptides were all terminated by a lysine residue while C‐terminal peptides did not contain lysine residue.…”

Section: Post‐translational Modification Proteomementioning

confidence: 99%

Section: Post‐translational Modification Proteomementioning

confidence: 99%

Discover the Post‐Translational Modification Proteome Using Mass Spectrometry

et al. 2021

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Protein post‐translational modifications (PTMs) are chemical modifications on proteins. PTMs play a key role in many cellular processes by influencing the structure of proteins and dynamically regulating their functions. Therefore, characterizing PTMs at proteome level is critical to provide invaluable insight into the functions of proteins underlying different biological processes. Advances in modern proteomics technologies including sample preparation, chromatography separation as well as mass spectrometry have propelled the PTMs proteome to further depths. During the past decade, to better examine the PTMs with high sensitivity and selectivity, our group have developed a series of MS‐based novel analytical approaches to study the protein PTMs. Herein, we mainly introduce these approaches developed by our group and discuss how to overcome the technical obstacles of studying various protein PTMs with mass spectrometry. What is the most favorite and original method developed in your research group? The qualitative and quantitative approaches developed for analysis of PTM proteome. How do you get into this specific field? Could you please share some experiences with our readers? I entered Fudan University in 2003, when the sequencing of human genome was just completed. At that time, the life science entered the post‐genomic era, the era of proteomics. My post‐doctoral work is mainly based on mass spectrometry analysis, which is the core tool for proteome research. Therefore, I began to develop mass spectrometry‐based method for proteome research since then. What is the most important personality for scientific research? Creative, persistence, and collaboration. What's your hobbies? Playing badminton.

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Separation methods for system‐wide profiling of protein terminome

Wang

2022

Proteomics

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Protein N‐ and C‐termini have specific biochemical properties and functions. They play vital roles in various biological processes, such as protein stability and localization. In addition, post‐translational modifications and proteolytic processing generate different proteoforms at protein termini. In recent years, terminomics has attracted significant attention, and numerous strategies have been developed to achieve high‐throughput and global terminomics analysis. This review summarizes the recent protein N‐termini and C‐termini enrichment methods and their application in different samples. We also look ahead further application of terminomics in profiling protease substrates and discovery of disease biomarkers and therapeutic targets.

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In-Depth Analysis of C Terminomes Based on LysC Digestion and Site-Selective Dimethylation

Cited by 16 publications

References 33 publications

One-step Isolation of Protein C-terminal Peptides from V8 Protease-digested Proteins by Metal Oxide-based Ligand Exchange Chromatography

One-step Isolation of Protein C-terminal Peptides from V8 Protease-digested Proteins by Metal Oxide-based Ligand Exchange Chromatography

Discover the Post‐Translational Modification Proteome Using Mass Spectrometry

Separation methods for system‐wide profiling of protein terminome

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