Methods In Mass Spectrometry

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Mass spectrometric methods for the analysis of protein structures are being improved, applied and tested in independent and collaborative research projects in proteomics. Projects in separation methods and data processing are in progress. In the past year, progress has been made on implementation of a computational environment for mass spectrometric proteomic data processing; isotope labeled fusion proteins as protein standards; quantification of identified and unidentified components in sets of complex liquid chromatography-high resolution mass spectrometry data; and multi-institutional collaborative studies on standards in proteomics. The processing and mining of mass spectrometric proteomic data requires a system allowing investigators desktop computer access to terabytes of data; the ability to repeatedly probe their data with emerging software tools; and the ability to transfer this data to public archival repositories. A versatile system has been implemented in LNT and will be shared with other proteomics groups in NIH. We sought to determine if biosynthetic concatenated labeled peptides (concatemers) are equivalent to whole labeled proteins as internal standards for isotope dilution mass spectrometry using selected reaction monitoring on a triple quadrupole mass analyzer. Mass spectrometry provides a platform for these measurements by using multiple reaction monitoring to follow specific transitions of peptides as they fragment; however, internal standards are only accurate if they faithfully mimic proteolytic properties of full-length proteins. We selected signature peptides plus 12 amino acids (6 amino- and 6 carboxy-terminal) through mass spectrometric screening as well as the public databases and literature. Synthetic genes for the extended selected sequences are fused with affinity tags and expressed by cloning a synthetic gene into an expression vector and labeled using 13C and 15N arginine and lysine amino acids. A human serum albumin (HSA) concatemer was tested because native HSA is readily available as well as 15N-labeled full-length HSA as a laboratory standard. HSA concatemer concentration was measured with respect to a chemically synthesized strep tag 10-mer peptide using a standard curve. Time, temperature, and enzyme studies were optimized. Three of the five peptides in the HSA concatemer accurately mimic the tryptic properties of native HSA. We demonstrated that concatenated HSA peptides can be used as internal standards for the quantification of HSA in urine samples. Traditional immunoturbidimetric data provided comparable results. For the immunoturbidimetric assays, 50 microliters of urine are consumed; for the selected reaction monitoring mass spectrometry method, less than 5 microliters are consumed.