title: Microbore liquid chromatography ultraviolet detection for quantification of total peptide amount and its application for assessing sample quality in shotgun proteome analysis of hundreds of cells.
abstract: Mass spectrometric profiling of the proteome of a small number of cells requires not only a sensitive instrument for protein/peptide separation and detection, but also a robust sample preparation protocol to process a very small amount of proteins (<1μg) present in few cells. We have developed and evaluated the performance of a microbore liquid chromatography (LC) UV detection system for quantifying the total amount of peptides in a shotgun proteome analysis workflow that is tailored for the analysis of hundreds of cancer cells. Upon the sample injection into a 1-mm-diameter reversed phase column, a step-gradient was used to first remove salts and other impurities and then elute the peptides quickly without much separation. The UV absorbance of eluted peptides at 214nm was used for peptide quantification with the aid of a calibration curve of a tryptic digest of a mixture of four standard proteins. Two linear calibration regions could be obtained in the peptide amount ranging from 0.03μg to 0.3μg and from 0.6μg to 5μg. The limit of quantification (LOQ) was determined to be 30ng (or 39ng in the linear calibration range). However, the presence of background proteins, mainly keratins, introduced during the sample preparation process was found to be the limiting factor in quantifying a lower amount of peptides from a cell lysate digest. With background absorbance from the digest of contaminant proteins in a solution, the LOQ was found to be 200ng. This nondestructive microbore LC-UV method should be useful in assessing sample quality during the development and applications of an efficient sample preparation method for proteome analysis of a small number of cells. As an example, this method was used for quantifying the peptides generated from breast cancer MCF-7 cell extracts with a limited number of cells: 250, 500 and 1000 cells. Using capillary LC quadrupole time-of-flight mass spectrometry, 81-126, 122-154 and 256-282 proteins could be identified from 250, 500, and 1000 cells, respectively, in duplicate experiments. This method was also applied for the analysis of biological triplicate samples of MCF-7 cells. The average numbers of peptides and proteins detected from the experimental triplicate analyses of biological triplicate samples were 400±71 (9 datasets) and 124±14, respectively, from 250 cells, and 531±44 and 162±16, respectively, from 500 cells.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Yanan
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Lu
authors.initials: L
authors.lastname: Chen
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada. Electronic address: Liang.Li@ualberta.ca.
authors.forename: Liang
authors.initials: L
authors.lastname: Li
mesh_terms: D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D061986:MCF-7 Cells; D010455:Peptides / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D014466:Ultraviolet Rays
chemical_list: D010455:Peptides; D020543:Proteome
keywords: Liquid chromatography; Mass spectrometry; Peptide quantification; Protein identification; Proteomics; UV detection