Quantitative proteomic analysis using isobaric protein tags enables rapid comparison of changes in transcript and protein levels in transformed cells.
Isobaric tags for relative and absolute quantitation, an approach to concurrent, relative quantification of proteins present in four cell preparations, have recently been described. To validate this approach using complex mammalian cell samples that show subtle differences in protein levels, a model stem cell-like cell line (FDCP-mix) in the presence or absence of the leukemogenic oncogene TEL/PDGFRbeta has been studied. Cell lysates were proteolytically digested, and peptides within each sample were labeled with one of four isobaric, isotope-coded tags via their N-terminal and/or lysine side chains. The four labeled samples are mixed and peptides separated by two-dimensional liquid chromatography online to a mass spectrometer (LC-MS). Upon peptide fragmentation, each tag releases a distinct mass reporter ion; the ratio of the four reporters therefore gives relative abundances of the given peptide. Relative quantification of proteins is derived using summed data from a number of peptides. TEL/PDGFRbeta leukemic oncogene-mediated changes in protein levels were compared with those seen in microarray analysis of control and transfected FDCP-mix cells. Changes at the protein level in most cases reflected those seen at the transcriptome level. Nonetheless, novel differences in protein expression were found that indicate potential mechanisms for effects of this oncogene.
Unwin
Richard D
RD
Department of Faculty of Medical and Human Sciences, University of Manchester, Christie Hospital, Withington, Manchester, M20 9BX, United Kingdom.
Pierce
Andrew
A
Watson
Rod B
RB
Sternberg
David W
DW
Whetton
Anthony D
AD
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000378:metabolism* / Q000473:pathology; D002853:Chromatography, Liquid; D015972:Gene Expression Regulation, Neoplastic; D006412:Hematopoietic Stem Cells / Q000378:metabolism / Q000473:pathology; D007553:Isotope Labeling; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D039902:Multipotent Stem Cells / Q000378:metabolism / Q000473:pathology; D015514:Oncogene Proteins, Fusion / Q000096:biosynthesis / Q000235:genetics; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D014158:Transcription, Genetic
D015514:Oncogene Proteins, Fusion; D010455:Peptides; D020543:Proteome; C120888:TEL-PDGFRbeta fusion protein, human; D008239:Lysine