title: Automation of dimethylation after guanidination labeling chemistry and its compatibility with common buffers and surfactants for mass spectrometry-based shotgun quantitative proteome analysis.
abstract: Isotope labeling liquid chromatography-mass spectrometry (LC-MS) is a major analytical platform for quantitative proteome analysis. Incorporation of isotopes used to distinguish samples plays a critical role in the success of this strategy. In this work, we optimized and automated a chemical derivatization protocol (dimethylation after guanidination, 2MEGA) to increase the labeling reproducibility and reduce human intervention. We also evaluated the reagent compatibility of this protocol to handle biological samples in different types of buffers and surfactants. A commercially available liquid handler was used for reagent dispensation to minimize analyst intervention and at least twenty protein digest samples could be prepared in a single run. Different front-end sample preparation methods for protein solubilization (SDS, urea, Rapigest™, and ProteaseMAX™) and two commercially available cell lysis buffers were evaluated for compatibility with the automated protocol. It was found that better than 94% desired labeling could be obtained in all conditions studied except urea, where the rate was reduced to about 92% due to carbamylation on the peptide amines. This work illustrates the automated 2MEGA labeling process can be used to handle a wide range of protein samples containing various reagents that are often encountered in protein sample preparation for quantitative proteome analysis.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Andy
authors.initials: A
authors.lastname: Lo
authors.forename: Yanan
authors.initials: Y
authors.lastname: Tang
authors.forename: Lu
authors.initials: L
authors.lastname: Chen
authors.forename: Liang
authors.initials: L
authors.lastname: Li
mesh_terms: D001331:Automation; D002021:Buffers; D004867:Equipment Design; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D008745:Methylation; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry; D014508:Urea / Q000737:chemistry
chemical_list: D002021:Buffers; D029968:Escherichia coli Proteins; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D019791:Guanidine
keywords: Chemical derivatization; Isotope labeling; Labeling efficiency; Liquid chromatography–mass spectrometry; Proteomics