Matrix-assisted laser desorption/ionization-MS-based relative quantification of peptides and proteins using iodoacetamide and N-methyliodoacetamide as labeling reagents.
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.
Article: Evaluating the Potential Nonthermal Microwave Effects of Microwave-Assisted Proteolytic Reactions.[show abstract] [hide abstract]
ABSTRACT: Microwave-assisted proteolytic digestion methods have evolved into a highly effective approach and serve as an alternative to conventional overnight digestion. This approach typically exploits the unique microwave properties to facilitate the digestion of proteins into their peptides within minutes. Conventional digestion is carried out at 37°C while microwave-assisted digestion requires much higher and sometimes inconsistent temperatures. Thus, this study aims to investigate whether the faster reaction rate is due to the microwave quantum effect or the thermal effect. Quantitative mass spectrometry was used to conduct kinetic analysis of tryptic digestion for several proteins by microwave and conventional heating. The percentages of digestion products relative to internal standards showed no significant difference between microwave and conventional heating conditions at the same digestion temperature. The optimum temperature for trypic digestion was determined to be 50°C. Furthermore, this study compares the digestion completeness indicators of several proteins under microwave and conventional heating. Again, the values obtained from microwave and conventional heating were similar given identical temperatures. The overall results prove that a nonthermal effect does not exist in microwave-assisted tryptic digestion. Therefore, conventional heating at high temperatures (50°C) can be also used to accelerate digestion reactions.Journal of proteomics 01/2013; · 5.07 Impact Factor