ABSTRACT
The use of iodoacetamide (IAA) and N-methyliodoacetamide (MIAA) as labeling agents for the relative measurements of proteins using MALDI-MS is described herein. These reagents, which alkylate the thiol groups of cysteine residues in proteins, were introduced during the alkylation step of a common protein denaturation and digestion process. This approach is simpler and cheaper than those involving isotope labeling agents. The labeling agents described herein displayed good dynamic ranges and correlation coefficients for protein quantification analyses when the proteins were treated through either in-solution or in-gel digestion. The best dynamic ranges (in the molar ratio) for proteins lysozyme, transferrin, and BSA (in-solution digestion) are 0.1-10, 0.1-8, and 0.1-8, respectively. The corresponding correlation coefficients are greater than 0.99. The IAA/MIAA labeling is a useful method for the relative quantification of peptides and digested proteins when the chromatographic isotope effect is not a major concern.
Subject(s)
Iodoacetamide/analogs & derivatives , Iodoacetamide/chemistry , Peptide Fragments/analysis , Proteins/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Amino Acid Sequence , Animals , Indicators and Reagents/chemistry , Muramidase/analysis , Muramidase/chemistry , Proteins/chemistry , Reproducibility of Results , Sensitivity and Specificity , Serum Albumin, Bovine/analysis , Serum Albumin, Bovine/chemistry , Transferrin/analysis , Transferrin/chemistryABSTRACT
The work describes the accelerated enzymatic digestion of several proteins in various solvent systems under microwave irradiation. The tryptic fragments of the proteins were analyzed by matrix-assisted laser desorption/ionization mass spectrometry. Under the influence of rapid microwave heating, these enzymatic reactions can proceed in a solvent such as chloroform, which, under traditional digestion conditions, renders the enzyme inactive. The digestion efficiencies and sequence coverages were increased when the trypsin digestions occurred in acetonitrile-, methanol- and chloroform-containing solutions that were heated under microwave irradiation for 10 min using a commercial microwave applicator. The percentage of the protein digested under microwave irradiation increased with the relative acetonitrile content, but decreased as the methanol content was increased. These observations suggest that acetonitrile does not deactivate the enzyme during the irradiation period; in contrast, methanol does deactivate it. In all cases, the digestion efficiencies under microwave irradiation exceed those under conventional conditions.
