Open AccessEvaluation of Strong Cation Exchange versus Isoelectric Focusing of Peptides for Multidimensional Liquid Chromatography-Tandem Mass Spectrometry
Author(s) -
Robbert J.C. Slebos,
Jonathan W. C. Brock,
Nancy F. Winters,
Sarah R. Stuart,
Misti A. Martinez,
Ming Li,
Mathew C. Chambers,
Lisa J. Zimmerman,
Amy Joan L. Ham,
David L. Tabb,
Daniel C. Liebler
Publication year - 2008
Publication title -
journal of proteome research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.644
H-Index - 161
eISSN - 1535-3907
pISSN - 1535-3893
DOI - 10.1021/pr8004666
Subject(s) - chemistry , proteome , chromatography , shotgun proteomics , tandem mass spectrometry , peptide , isoelectric focusing , shotgun , mass spectrometry , proteomics , biomarker discovery , peptide mass fingerprinting , bottom up proteomics , protein mass spectrometry , biochemistry , enzyme , gene
Shotgun proteome analysis platforms based on multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a powerful means to discover biomarker candidates in tissue specimens. Analysis platforms must balance sensitivity for peptide detection, reproducibility of detected peptide inventories and analytical throughput for protein amounts commonly present in tissue biospecimens (< 100 microg), such that platform stability is sufficient to detect modest changes in complex proteomes. We compared shotgun proteomics platforms by analyzing tryptic digests of whole cell and tissue proteomes using strong cation exchange (SCX) and isoelectric focusing (IEF) separations of peptides prior to LC-MS/MS analysis on a LTQ-Orbitrap hybrid instrument. IEF separations provided superior reproducibility and resolution for peptide fractionation from samples corresponding to both large (100 microg) and small (10 microg) protein inputs. SCX generated more peptide and protein identifications than did IEF with small (10 microg) samples, whereas the two platforms yielded similar numbers of identifications with large (100 microg) samples. In nine replicate analyses of tryptic peptides from 50 microg colon adenocarcinoma protein, overlap in protein detection by the two platforms was 77% of all proteins detected by both methods combined. IEF more quickly approached maximal detection, with 90% of IEF-detectable medium abundance proteins (those detected with a total of 3-4 peptides) detected within three replicate analyses. In contrast, the SCX platform required six replicates to detect 90% of SCX-detectable medium abundance proteins. High reproducibility and efficient resolution of IEF peptide separations make the IEF platform superior to the SCX platform for biomarker discovery via shotgun proteomic analyses of tissue specimens.