Evaluation of sample fractionation using micro-scale liquid-phase isoelectric focusing on mass spectrometric identification and quantitation of proteins in a SILAC experiment.
Mass spectrometric methods based on stable isotopes have shown great promise for identification and quantitation of complex mixtures. Stable isotope labelling by amino acids in cell culture (SILAC) is a straightforward and accurate procedure for quantitation of proteins from cell lines, that are cultured in media containing the natural amino acid or its isotopically labelled analogue, giving rise to either 'light' or 'heavy' proteins. The two cell populations are pooled and treated as a single sample, which allows the use of various protein purification methods without introducing errors into the quantitative analysis. The quantitation of the proteins is based on the intensities of the light and heavy peptides. The increased number of peptides in a quantitative experiment arising from peptide pairs implies that prefractionation is critical prior to liquid chromatography/mass spectrometric (LC/MS) analysis to minimise signal suppression effects and errors in measurements of the intensity ratios. In this study, the effect of a prefractionation step on identification and quantitation of proteins in a SILAC experiment was evaluated. We show that micro-scale liquid-phase isoelectric focusing in the Micro Rotofor separates proteins into well-defined fractions and reduces the sample complexity. Furthermore, the fractionation enhanced the number of identified proteins and improved their quantitation.
Thorsell
Annika
A
Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Göteborg University, Sahlgrenska University Hospital, Mölndal, Sweden.
Portelius
Erik
E
Blennow
Kaj
K
Westman-Brinkmalm
Ann
A
D001120:Arginine / Q000378:metabolism; D045744:Cell Line, Tumor; D005591:Chemical Fractionation / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D005909:Glioblastoma / Q000378:metabolism; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry; D006801:Humans; D007525:Isoelectric Focusing; D007553:Isotope Labeling; D034882:Lamins / Q000737:chemistry; D008832:Microchemistry / Q000379:methods*; D008840:Microfilament Proteins / Q000737:chemistry; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000737:chemistry*; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D034882:Lamins; D008840:Microfilament Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D010455:Peptides; C071058:moesin; D001120:Arginine