Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

Author(s)

Correa, Claudia Neves ^{[1, 2]} ; Fiametti, Louise Oliveira ^[1] ; Mazzi Esquinca, Maria Eduarda ^[1] ; de Castro, Leandro Mantovani ^{[1, 2]}

Affiliation

^[1] Sao Paulo State Univ UNESP, Biosci Inst, Dept Biol & Environm Sci, Presidente Prudente, SP - Brazil

^[2] Sao Paulo State Univ UNESP, Biodivers Coastal Environm Postgrad Program, Biosci Inst, Presidente Prudente, SP - Brazil

• Document type: Journal article
• Source: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS; n. 177 NOV 2021.
• Web of Science Citations: 0

Abstract

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized. Peptidomic studies frequently analyze peptides that are naturally generated in cells. Here, a sample preparation protocol based on heat-inactivation is described, which eliminates protease activity, and extraction with mild conditions, so there is no peptide bonds cleavage. In addition, the relative quantitation of peptides using stable isotope labeling by reductive methylation of amines is also shown. This labeling method has some advantages as the reagents are commercially available, inexpensive compared to others, chemically stable, and allows the analysis of up to five samples in a single LC-MS run. (AU)

• FAPESP's process: 21/01286-1 - Tentacle transcriptome and peptidome of Bunodosoma caissarum sea anemone
• Grantee: Maria Eduarda Mazzi Esquinca
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 19/17433-3 - Evaluation of cytotoxic or neuroprotective effects of altered peptides on the central nervous system of zebrafish (Danio rerio) in Parkinson's Disease
• Grantee: Louise Oliveira Fiametti
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 19/16023-6 - Revealing transcriptome and proteome data for characterization of peptidomes and bioactive peptides in organisms from bycatch of shrimp trawling
• Grantee: Leandro Mantovani de Castro
• Support Opportunities: Regular Research Grants