Toward high sequence coverage of proteins in human breast cancer cells using on‐line monolith‐based HPLC‐ESI‐TOF MS compared to CE MS

Abstract

A method is developed toward high sequence coverage of proteins isolated from human breast cancer MCF10 cell lines using a 2-D liquid separations. Monolithic-capillary columns prepared by copolymerizing styrene with divinylbenzene are used to achieve highresolution separation of peptides from protein digests. This separation is performed with minimal sample preparation directly from the 2-D liquid fractionation of the cell lysate. The monolithic column separation is directly interfaced to ESI-TOF MS to obtain a peptide map. The protein digests were also analyzed by MALDI-TOF MS and an accurate M r of the intact protein was obtained using an HPLC-ESI-TOF MS. The result is that these techniques provide complementary information where nearly complete sequence coverage of the protein is obtained and can be compared to the experimental M r value. The high sequence coverage provides information on isoforms and other post-translational modifications that would not be available from methods that result in low sequence coverage. The results from the use of monolithic columns are compared to that obtained by CE-MS. The monolithic column separations provide a rugged and highly reproducible method for separating protein digests prior to MS analysis and is suited to confidently identify biomarkers associated with cancer progression.

Capillary electrophoresis‐electrospray‐mass spectrometry in peptide analysis and peptidomics

“…One of the main uses of CE-MS in proteomics/peptidomics research is protein identification through the analysis of the peptides produced after enzymatic digestion of the biopolymer [101], including the study of protein modifications [102]. In this regard, an important limitation arises from the frequent generation of unexpected peptidic fragments during the enzymatic digestion of any protein.…”

Monoliths in capillary electrochromatography and capillary liquid chromatography in conjunction with mass spectrometry

“…The method was developed toward high sequence coverage of proteins originated from a cancer cell lysate, which was subjected to a preparative liquid‐phase IEF in a first step of the whole protocol. Each obtained fraction was separated on an analytical particle packed column and then the individual fractions were digested and analyzed on a PS‐DVB capillary . The suggested HPLC‐ESI‐TOF‐MS method was robust and provided improved loadability compared to a tested CE‐MS method, which was subsequently used for the analysis of the digested peptides.…”

Proteins and Molecular Pathways Relevant for the Malignant Properties of Tumor-Initiating Pancreatic Cancer Cells

“…In order to enlarge this knowledge and to identify novel drug targets, performing omics experiments is a powerful strategy for the generation of comprehensive data sets for the unbiased study of molecular and functional differences between selected tumor cell populations [ 32 , 33 , 34 , 35 , 36 ]. Despite the broad range of CSC markers, comparison of different CSC populations in the studies mentioned above showed only limited overlap between the populations.…”