Acid hydrolysis of proteins in matrix assisted laser desorption ionization matrices.
Sample preparation is crucial to the success of experiments in biological mass spectrometry. In proteomics, digestion of the proteins into peptides is a key step for "bottom-up" approaches. Often, the use of enzymes requires physiological conditions, producing peptides that must be extracted or further purified before mass analysis. Chemical cleavage reagents offer more flexibility and can be more compatible with downstream mass analysis. Expanding on prior work using acid hydrolysis, proteolysis with matrix-assisted laser desorption ionization (MALDI) matrices is presented. This sample preparation can be performed rapidly with a minimum of reagents and sample handling, but it must first be evaluated in terms of digestion efficiency, missed cleavages, and side reactions before implementation for in-gel digestion and in-solution digestion using minimal volumes of protein. Time courses of acid hydrolysis are shown for protein standards, illustrating the sensitivity of this type of sample preparation, minimization of side reactions, and performance for proteins in mixtures. To illustrate the potential for sensitive detection of a specific protein, MALDI matrix hydrolysis is used to digest a protein immunoprecipitated from cell lysate.
Remily-Wood
Elizabeth
E
Molecular Oncology and Proteomics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612, USA.
Dirscherl
Hayley
H
Koomen
John M
JM
D000143:Acids / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002645:Chickens; D005240:Feasibility Studies; D005782:Gels / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000032:analysis / Q000737:chemistry; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics; D012015:Reference Standards; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014168:Transferrin / Q000032:analysis / Q000737:chemistry; D025801:Ubiquitin / Q000032:analysis / Q000737:chemistry
D000143:Acids; D005782:Gels; D010455:Peptides; D011506:Proteins; D014168:Transferrin; D025801:Ubiquitin; D010047:Ovalbumin
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