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

Summary

This article describes a sample preparation method based on heat-inactivation to preserve endogenous peptides avoiding degradation post-mortem, followed by relative quantitation using isotopic labeling plus LC-MS.

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.

Introduction

"Omics" sciences are characterized by the deep analysis of a molecule set, such as DNA, RNA, proteins, peptides, metabolites, etc. These generated large-scale datasets (genomics, transcriptomics, proteomics, peptidomics, metabolomics, etc.) have revolutionized biology and led to an advanced understanding of biological processes¹. The term peptidomics began to be introduced in the early 20^th century, and some authors have referred to it as a branch of proteomics². However, peptidomics has distinct particularities, where the main interest is to investigate the naturally generated peptides content during ....

Protocol

The following procedure for peptide extraction and reductive methylation was adapted from previously published procedures^24,25,26,27. This protocol followed the guidelines of the National Council for Animal Experimentation Control (CONCEA) and was approved by the Ethics Commission for Animal Use (CEUA) at Bioscience Institute of Sao Paulo State University. The protocol steps are shown in

Discussion

In most peptidomics studies, one of the critical steps is, without doubt, the sample preparation that should be carefully performed to avoid the presence of peptide fragments generated by proteases after a few minutes post-mortem. The initial studies on brain extracts prepared from non-microwaved samples showed a large number of protein fragments to be present in the 10-kDa microfiltrates. Different approaches have been described to avoid peptide spectra from protein degradation: focused microwave irradiation animal sacr.......

Materials

Name	Company	Catalog Number	Comments
10 kDa cut-off filters	Merck Millipore	UFC801024	Amicon Ultra-4, PLGC Ultracel-PL Membrane, 10 kDa
Acetone	Sigma-Aldrich	179124
Acetonitrile	Sigma-Aldrich	1000291000
Ammonium bicarbonate	Sigma-Aldrich	11213
analytical column (EASY-Column)	EASY-Column	(SC200)	10 cm, ID75 µm, 3 µm, C18-A2
Ethyl 3-aminobenzoate methanesulfonate	Sigma-Aldrich	E10521	MS-222
Fluorescamine	Sigma-Aldrich	F9015
Formaldehyde solution	Sigma-Aldrich	252549
Formaldehyde-13C, d2, solution	Sigma-Aldrich	596388
Formaldehyde-d2 solution	Sigma-Aldrich	492620
Formic acid	Sigma-Aldrich	33015
Fume hood	Quimis	Q216
Hydrochloric acid - HCl	Sigma-Aldrich	258148
LoBind-Protein retention tubes	Eppendorf	EP0030108116-100EA
LTQ-Orbitrap Velos	Thermo Fisher Scientific	LTQ Velos
Microwave oven	Panasonic	NN-ST67HSRU
n Easy-nLC II nanoHPLC	Thermo Fisher Scientific	LC140
PEAKS Studio	Bioinformatics Solutions Inc.	VERSION 8.5
Phosphate-buffered saline	Invitrogen	3002	tablets
precolumn (EASY-Column)	Thermo Fisher Scientific	(SC001)	2 cm, ID100 µm, 5 µm, C18-A1
Refrigerated centrifuge	Hermle	Z326K	for conical tubes
Refrigerated centrifuge	Vision	VS15000CFNII	for microtubes
Reversed-phase cleanup columns   (Oasis HLB 1 cc Cartridge)	Waters	186000383	Oasis HLB 1 cc Cartridge
Sodium cyanoborodeuteride - NaBD3CN	Sigma-Aldrich	190020
Sodium cyanoborohydride - NaBH3CN	Sigma-Aldrich	156159
Sodium phosphate dibasic	Sigma-Aldrich	S9763	NOTE: 0.2 M PB= 0.1 M phosphate buffer pH 6.8 (26.85 mL of Na2HPO3 1M) plus 0.1 M phosphate buffer pH 6.8 (23.15 mL of NaH2PO3 1M) to 250 ml of water
Sodium phosphate monobasic	Sigma-Aldrich	S3139
Sonicator	Qsonica	Q55-110
Standard peptide	Proteimax		amino acid sequence: LTLRTKL
Triethylammonium buffer - TEAB 1 M	Sigma-Aldrich	T7408
Trifluoroacetic acid - TFA	Sigma-Aldrich	T6508
Ultra purified water	Milli-Q	Direct-Q 3UV
Vacuum centrifuge	GeneVac	MiVac DNA concentrator
Water bath	Cientec	266
Xcalibur Software	ThermoFisher Scientific	OPTON-30965