Investigation of methods suitable for the matrix-assisted laser desorption/ionization mass spectrometric analysis of proteins from ribonucleoprotein complexes.
A variety of protein isolation and purification techniques for ribonucleoprotein (RNP) complexes were investigated for their compatibility with downstream analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Ribosomal proteins from Escherichia coli 70S ribosomes were obtained using methods such as phenol extraction and precipitation by organic solvents or acids. Under optimal conditions, more than 90% of the expected ribosomal proteins were detected in a single MALDI-MS experiment. The most effective approach combined ribosome denaturation by buffer exchange with acid precipitation of the ribosomal ribonucleic acids. An improved acid precipitation approach, involving the sequential additions of acetic and trifluoroacetic acid, yielded more complete protein coverage while minimizing loss of ion signal from lower molecular weight proteins. With phenol extraction, substantial gains in ion abundance of higher molecular weight proteins are noted, although some of the lower molecular weight proteins were not efficiently extracted. These results illustrate several effective approaches for protein isolation from protein complexes such as RNPs that are MALDI-MS compatible, and these approaches should extend the use of MALDI-MS for proteomics-based analyses of other protein-nucleic acid complexes.
Suh
Moo-Jin
MJ
Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, PO Box 210172, University of Cincinnati, Cincinnati, OH 45221, USA.
Limbach
Patrick A
PA
D000096:Acetone; D000595:Amino Acid Sequence; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D019800:Phenol; D012261:Ribonucleoproteins / Q000032:analysis* / Q000737:chemistry*; D012269:Ribosomal Proteins / Q000032:analysis / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D046911:Macromolecular Substances; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins; C015494:ribosomal protein S1; D000096:Acetone; D019800:Phenol; D000431:Ethanol