Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Summary

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

Abstract

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Introduction

Measuring concentration differences of many proteins between complex samples is a central challenge in proteomics. Increasingly, this is being done by labeling proteins in each sample with different isotopic tags, combining the samples, and using mass spectrometry to quantify concentration differences. Several methods exist for stable isotopic labeling of proteins and peptides. ¹⁵N labeling¹ and SILAC² introduce isotopic labels metabolically in vivo, whereas iCAT³, iTRAQ⁴, and reduction dimethylation⁵ add stable isotope tags after protein extraction and digestion. Among these methods, reductive....

Protocol

NOTE: This method was previously described¹².

1. Protein Isolation

Prepare 1 mg of cellular protein by lysing cells, preferably by physical methods such as French press, bead beating, or sonication. Avoid lysozyme-mediated cell lysis because the enzyme will confound mass spectrometry measurements.

2. TCA Precipitation of Proteins

Add 1 volume trichloroacetic acid (TCA) to 4 volumes protein and chill on i.......

Discussion

Several points make stable isotope labeling of peptides using reductive dimethylation (ReDi labeling) an attractive method for quantitative proteomics: inexpensive labeling reagents (reagents cost less than $1 per sample), fast reaction rate (~10 min), absence of side products, high reproducibility (Figures 3, 4), stable reaction products, ability to use any protease, and high ionization efficiency of labeled peptides. Chemical labeling by ReDi is also advantageous relative to metabolic labeling sin.......

Materials

Name	Company	Catalog Number	Comments
trichloroacetic acid	Sigma-Aldrich	T9159	protein precipitation
acetone	Sigma-Aldrich	650501	protein precipitation
Sodium dodecyl sulfate	Sigma-Aldrich	71736	denature, reduce protein
sodium hydroxide	Sigma-Aldrich	S8045	denature, reduce protein
DL-Dithiothreitol	Sigma-Aldrich	43816	denature, reduce, alkylate protein
protease Inhibitor Complete Mini Cocktail	Roche	4693124001	denature, reduce protein
iodoacetamide	Sigma-Aldrich	I6125	alkylate protein
HEPES	Sigma-Aldrich	H7523	resuspend, extract, label protein
calcium chloride	Sigma-Aldrich	C5670	resuspend protein
Lysyl Endoprotease	Wako Chemicals	129-02541	protein digestion
sequencing grade trypsin	Promega	V5111	protein digestion
acetic acid	Sigma-Aldrich	320099	protein digestion
trifluoroacetic acid	Sigma-Aldrich	299537	Reversed-phase peptide extraction
tC18 Sep-Pak C18 cartridges	Waters	WAT054960	Reversed-phase peptide extraction
extraction manifold	Waters	WAT200609	Reversed-phase peptide extraction
acetonitrile	Sigma-Aldrich	14261	various
formaldehyde	Sigma-Aldrich	252549	“light” peptide labeling
cyanoborohydride	Sigma-Aldrich	71435	“light” peptide labeling
deuterated formaldehyde	Sigma-Aldrich	492620	“heavy” peptide labeling
sodium cyanoborodeuteride	CDN isotopes	D-1797	“heavy” peptide labeling
MES	Sigma-Aldrich	M3671	peptide labeling
C18-HPLC column (4.6 x 250 mm, 5 µm particle size)	Agilent	770450-902	basic pH reversed-phase chromatography
formic acid	Sigma-Aldrich	399388	various
C18 Empore Disks	3M	14-386-3	STAGE tips
methanol	Sigma-Aldrich	494437	STAGE tips