Quantitative Analysis of Snake Venoms Using Soluble Polymer-based Isotope Labeling

Jacob A Galan,W Andy Tao,Alexis Rodriguez-Acosta,Minjie Guo,Elda E Sanchez,John C Perez,Esteban Cantu

doi:10.1074/mcp.m700321-mcp200

Abstract

We present the design and synthesis of a new quantitative strategy termed soluble polymer-based isotope labeling (SoPIL) and its application as a novel and inclusive method for the identification and relative quantification of individual proteins in complex snake venoms. The SoPIL reagent selectively captures and isolates cysteine-containing peptides, and the subsequent tagged peptides are released and analyzed using nanoflow liquid chromatography-tandem mass spectrometry. The SoPIL strategy was used to quantify venom proteins from two pairs of venomous snakes: Crotalus scutulatus scutulatus type A, C. scutulatus scutulatus type B, Crotalus oreganus helleri, and Bothrops colombiensis. The hemorrhagic, hemolytic, clotting ability, and fibrinogenolytic activities of crude venoms were measured and correlated with difference in protein abundance determined by the SoPIL analysis. The SoPIL approach could provide an efficient and widely applicable tool for quantitative proteomics.

Highlights

We present the design and synthesis of a new quantitative strategy termed soluble polymer-based isotope labeling (SoPIL) and its application as a novel and inclusive method for the identification and relative quantification of individual proteins in complex snake venoms
Current methods either include extra purification steps that could lead to severe sample loss or solid phase extraction that is usually limited by heterogeneous reaction and nonlinear kinetics
This study presents the first quantitative proteomics analysis of snake venom from several species based on stable isotope labeling

Summary

Introduction

We present the design and synthesis of a new quantitative strategy termed soluble polymer-based isotope labeling (SoPIL) and its application as a novel and inclusive method for the identification and relative quantification of individual proteins in complex snake venoms. The traditional and frequently used method to investigate differential protein abundances on a large scale between samples from different sources is the staining of proteins separated by two-dimensional (2D) PAGE. The heterogeneous nature of solid phase reaction for proteomics research presents a serious issue for sample recovery and identification, which usually deal with a small amount of proteins and peptides in extremely complex mixtures.

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