Dataset Information

Development of sequential sample processing protocol on a single anion-exchange solid-phase extraction disc for bottom-up/cross-linking mass spectrometry

ABSTRACT: In this project, we developed a rapid enzymatic digestion protocol within an anion exchange StageTip named AnExSP (anion-exchange disc-assisted sequential sample preparation) and, using data-independent acquisition, analyzed its digestion performance of human cellular protein extracts (HCPE) using trypsin and trypsin/Lys-C. The outstanding effectiveness and simplicity of AnExSP in sample preparation for gel-based cross-linking mass spectrometry was also verified.

ORGANISM(S): Homo sapiens

SUBMITTER: Nobuaki Takemori

PROVIDER: PXD029444 | JPOST Repository | Wed Apr 20 00:00:00 BST 2022

REPOSITORIES: jPOST

ACCESS DATA

Publications

Bottom-up/cross-linking mass spectrometry <i>via</i> simplified sample processing on anion-exchange solid-phase extraction spin column.

Takemori Ayako A Kawashima Yusuke Y Takemori Nobuaki N

Chemical communications (Cambridge, England) 20220118 6

We introduce a simple single-column protein digestion method for low-microgram-level samples containing sodium dodecyl sulfate and Coomassie dye that can be completed within a few hours. ...[more]

PMID: 34897310

Similar Datasets

Project description:Sample preparation is a critical process for proteomic studies. Many efficient and reproducible sample preparation methods have been developed for mass spectrometry-based proteomic analysis of human and animal tissues or cells, but no attempt has been made to evaluate these protocols for plants. We here present an LC-MS/MS-based proteomics study of barley leaf aimed at optimization of methods to achieve efficient and unbiased trypsin digestion of proteins prior to LC-MS/MS based sequencing and quantification of peptides. We evaluated two spin filter-aided sample preparation protocols using either sodium dodecyl-sulphate or sodium deoxycholate (SDC), and three in-solution digestion (ISD) protocols using SDC or trichloroacetic acid/acetone precipitation.The proteomics workflow identified and quantified up to 1800 barley proteins based on sequencing of up to 6900 peptides per sample. The two spin filter-based protocols provided a 12-38% higher efficiency than the ISD protocols, including more proteins of low abundance. Among the ISD protocols, a simple one-step reduction and S-alkylation method (OP-ISD) was the most efficient for barley leaf sample preparation; it identified and quantified 1500 proteins and displayed higher peptide-to-protein inference ratio and higher average amino acid sequence coverage of proteins. The two spin filter-aided sample preparation protocols are compatible with TMT labelling for quantitative proteomics studies. They exhibited complementary performance as about 30% of the proteins were identified by either one or the other protocol, but also demonstrated a positive bias for membrane proteins when using SDC as detergent.We provide detailed protocols for efficient plant protein sample preparation for LC-MS/MS-based proteomics studies. Spin filter-based protocols are the most efficient for the preparation of leaf samples for MS-based proteomics. However, a simple protocol provides comparable results although with different peptide digestion profile.

Dataset Information

Development of sequential sample processing protocol on a single anion-exchange solid-phase extraction disc for bottom-up/cross-linking mass spectrometry

Publications

Bottom-up/cross-linking mass spectrometry <i>via</i> simplified sample processing on anion-exchange solid-phase extraction spin column.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure