A combination of protein profiling and isotopomer analysis using matrix-assisted laser desorption/ionization-time of flight mass spectrometry reveals an active metabolism of the extracellular matrix of 3T3-L1 adipocytes.
Differential gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a commonly used protein profiling method. However, observed changes can be explained in multiple ways, one of which is by the protein turnover rate. In order to easily and rapidly obtain information on both the identity and turnover of individual proteins, we applied a combination of protein labeling with L-(ring-2,3,4,5,6 2H5) phenylalanine and MALDI-TOF MS. While the spectrum reveals the identity of a protein, mass isotopomer analysis provides information about the rate of protein labeling as a measure of synthesis or turnover. Using this approach on mature 3T3-L1 adipocytes, we were able to discriminate between rapidly and slowly metabolised proteins. In our isolate, proteins of the cytoskeleton appeared to be slowly metabolised, whereas components of the extracellular matrix, in particular collagen type I alpha 1 (COL1A1) and collagen type I alpha 2 (COL1A2) showed rapid accumulation of newly synthesized proteins. Both proteins appeared to be metabolised in the same ratio as they are present in collagen fibers, i.e. 2:1 (COL1A1: COL1A2). In addition, functionally related proteins were also readily labeled. Taken together, we have shown that a combination of stable isotope labeling and protein profiling by gel electrophoresis and MALDI-TOF analysis can simultaneously provide information on the identity and relative metabolic rate of proteins in eukaryotic cells in a simple, nonhazardous and rapid-throughput way.
Bouwman
Freek
F
Department of Human Biology, Nutrition and Toxicology Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands.
Renes
Johan
J
Mariman
Edwin
E
D041721:3T3-L1 Cells; D000595:Amino Acid Sequence; D000818:Animals; D003094:Collagen / Q000378:metabolism; D024042:Collagen Type I / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D003599:Cytoskeleton / Q000378:metabolism; D019992:Databases as Topic; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D020869:Gene Expression Profiling; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000494:pharmacology
C000716127:COL1A1 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D010455:Peptides; D003094:Collagen; D014357:Trypsin