The practice of quantifying proteins by peptide fragments from enzymatic proteolysis (digestion) was assessed regarding accuracy, reliability, and uncertainty of the results attainable. Purified recombinant growth hormone (rhGH, 22 kDa isoform) was used as a model analyte. Two tryptic peptides from hGH, T6 and T12, were chosen to determine the amount of the protein in the original sample. Reference solutions of T6 and T12 (isotopically labeled forms), value assigned by quantitative amino acid analysis (AAA) after complete hydrolysis, were used as internal standards. The accuracy of protein quantification by fragments T6 and T12 was evaluated by comparison of peptide results to those obtained for the same rhGH sample by AAA. The rate of cleavage (and thus the experimental protocol used) turned out to be crucial to the quality of results in protein quantification using enzymatic fragments. Applying a protocol customarily found in (qualitative) bottom-up proteomics gave results significantly higher than the target value from AAA (+11% with T6 and +6% with T12). In contrast, using a modified protocol optimized for fast and complete hydrolysis, results were unbiased within the limits of uncertainty, while the time needed for completion of proteolysis was considerably reduced (30 min as compared to 1080-1200 min). The method assessed highlighted three important criteria deemed necessary for successful protein quantification using proteolysis-based mass spectrometry methods. These are the following: the requirement for both the selected peptides and labeled internal standard to be stable throughout digestion; the correct purity assignment to the selected peptide standards; the proof of equimolar release of the selected peptides. The combined (overall) uncertainty for protein quantification was established by combination of estimates obtained for individual components and found to be U ) 4% for this example. This uncertainty is of the same order as that typically attainable in quantification of "small" organic molecules using liquid chromatography/isotope dilution mass spectrometry.The past decade has seen a significant increase in the development of mass spectrometric methods for protein quantification.1,2 Although it is viable to directly analyze intact proteins 3,4 by liquid chromatography/mass spectrometry (LC/MS), most of the examples of quantification that have been described are based on specific cleavage by enzymatic proteolysis (digestion) of the proteins down to smaller fragments, most of which are still long enough in amino acid sequence to provide specificity for the precursor protein, even in a complex mixture. [5][6][7][8][9][10][11][12][13][14] This enables the simplification of the quantification process to the analysis of short sequences of amino acids which are amenable to standard LC/MS techniques. Using isotopically labeled forms of the peptides as internal standards potentially introduces the advantages of reliability, accuracy, and repeatability into protein quantification that have been demonstrat...

A novel mechanism of monolayer poration leading to the rapid exfoliation and lysis of microbial membranes is reported.

No abstract