title: Combinatorial use of offline SCX and online RP-RP liquid chromatography for iTRAQ-based quantitative proteomics applications.
abstract: Extensive front-end separation is usually required for complex samples in bottom-up proteomics to alleviate the problem of peptide undersampling. Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-based experiments have particularly higher demands, in terms of the number of duty cycles and the sensitivity, to confidently quantify protein abundance. Strong cation exchange (SCX)/reverse phase (RP) liquid chromatography (LC) is currently used routinely to separate iTRAQ-labeled peptides because of its ability to simultaneously clean up the iTRAQ reagents and byproducts and provide first-dimension separation; nevertheless, the low resolution of SCX means that peptides can be redundantly sampled across fractions, leading to loss of usable duty cycles. In this study, we explored the combinatorial application of offline SCX fractionation with online RP-RP applied to iTRAQ-labeled chloroplast proteins to evaluate the effect of three-dimensional LC separation on the overall performance of the quantitative proteomics experiment. We found that the higher resolution of RP-RP can be harnessed to complement SCX-RP and increase the quality of protein identification and quantification, without significantly impacting instrument time and reproducibility.
authors.affiliation: Department of Chemistry, The University of Hong Kong, Hong Kong.
authors.forename: Edward
authors.initials: E
authors.lastname: Lau
authors.forename: Maggie P Y
authors.initials: MP
authors.lastname: Lam
authors.forename: S O
authors.initials: SO
authors.lastname: Siu
authors.forename: Ricky P W
authors.initials: RP
authors.lastname: Kong
authors.forename: Wai Lung
authors.initials: WL
authors.lastname: Chan
authors.forename: Zhongjun
authors.initials: Z
authors.lastname: Zhou
authors.forename: Jirong
authors.initials: J
authors.lastname: Huang
authors.forename: Clive
authors.initials: C
authors.lastname: Lo
authors.forename: Ivan K
authors.initials: IK
authors.lastname: Chu
mesh_terms: D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000032:analysis; D002412:Cations; D002736:Chloroplasts / Q000378:metabolism; D002852:Chromatography, Ion Exchange / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D029681:Arabidopsis Proteins; D002412:Cations; D010455:Peptides; D020543:Proteome