Quantitative Proteomic Analysis Using Isobaric Protein Tags Enables Rapid Comparison of Changes in Transcript and Protein Levels in Transformed Cells

Abstract

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/PDGFR␤ 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 twodimensional 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/PDGFR␤ 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. Molecular & Cellular Proteomics 4:924 -935, 2005.The development of approaches for measurement of relative expression of proteins between two (or more) samples is an essential aspect of systems biology. A common technique for this type of proteomic study has been two-dimensional gel electrophoresis, where proteins are separated by isoelectric point and molecular weight, and spot patterns are compared by sophisticated computer algorithms. Proteins of interest are then identified by MS. However, gel-based approaches have drawbacks, including amount of material required, reproducibility, and limiting of sensitivity by protein loss. These are balanced against recent advances in protein staining that allow intragel comparison of protein quantity from two or three samples (1).An alternative approach to proteomic analysis is LC-MS (2). This provides an increased sensitivity compared with gelbased approaches and can catalogue protein present in a sample. However, relative quantitation of proteins using LC-MS is challenging. Quantification by analyzing two samples in parallel and comparing their mass spectrometric profiles is not feasible. Isotopic labeling of peptides, however, does allow two samples to be analyzed in a single experiment. Isotope-coded affinity tagging using ICAT reagent technology indicates peptide source, with peak height giving relative quantity (3). Comparing the ICAT reagent approach with two-dimensional gel electrophoresis, however, demonstrates that neither offers comprehensive coverage of a proteome (4). This is true in part because many proteins (and therefore peptides) do not contain cysteine, the amino acid used for covalent attachment of...

A Statistical Model for iTRAQ Data Analysis

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