Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry

• Published in: Nature biotechnology Vol. 19; no. 10; pp. 946 - 951
• Main Authors: Aebersold, Ruedi, Han, David K, Eng, Jimmy, Zhou, Huilin
• Format: Journal Article
• Language: English
• Published: New York, NY Nature 01.10.2001; Nature Publishing Group
• Subjects: Affinity Labels; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Automation; Biological and medical sciences; Biotechnology; Cell Differentiation; Cells; Chromatography; Diverse techniques; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; HL-60 Cells; Humans; Leukemia; Mass Spectrometry; Microsomes - chemistry; Molecular and cellular biology; Molecular Sequence Data; Peptides; Proteins; Proteins - analysis; Proteomics; Publishing; Quantitative analysis; Reagents; Scientific imaging; Software; Software utilities; Tetradecanoylphorbol Acetate - pharmacology; Human; Promyelocytic leukemia; Multidimensional chromatography; Data analysis; Membrane protein; Isotope labelling; Identification; Method; Microsome; Cell line; Software; Mass spectrometry; Quantitative analysis
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/nbt1001-946

An approach to the systematic identification and quantification of the proteins contained in the microsomal fraction of cells is described. It consists of three steps: (1) preparation of microsomal fractions from cells or tissues representing different states; (2) covalent tagging of the proteins with isotope-coded affinity tag (ICAT) reagents followed by proteolysis of the combined labeled protein samples; and (3) isolation, identification, and quantification of the tagged peptides by multidimensional chromatography, automated tandem mass spectrometry, and computational analysis of the obtained data. The method was used to identify and determine the ratios of abundance of each of 491 proteins contained in the microsomal fractions of naïve and in vitro- differentiated human myeloid leukemia (HL-60) cells. The method and the new software tools to support it are well suited to the large-scale, quantitative analysis of membrane proteins and other classes of proteins that have been refractory to standard proteomics technology.