Ammonium bicarbonate buffer system for DNA hybridization and quantification by LC-ESI-MS/MS.

High salt buffer may be used for the UV/VIS or radiometric based detection of trihybrid DNA but would contaminate the electrospray mass spectrometer. Colorimetric DNA hybridization assays with the substrate BCIP/NBT reacted with the alkaline phosphatase-streptavidin (APSA) enzyme conjugate that showed a linear range for HIV DNA from 1 pM to 100 pM DNA in presence of high salt concentrations. Ammonia bicarbonate (AMBIC) or ethanolamine resulted in strong DNA hybridization similar to NaCl but was compatible with specific and sensitive mass spectrometry.

Selected Ion Extraction of Peptides with Heavy Isotopes and Hydrogen Loss Reduces the Type II Error in Plasma Proteomics.

Naturally occurring peptides display a wide mass distribution after ionization due to the presence of heavy isotopes of C, H, N, O, and S and hydrogen loss. There is a crucial need for sensitive methods that collect as much information as possible about all plasma peptide forms. Statistical analysis of the delta mass distribution of peptide precursors from MS/MS spectra that were matched to 63,077 peptide sequences by X!TANDEM revealed Gaussian peaks representing heavy isotopes and hydrogen loss at integer delta mass values of -3, -2, -1, 0, +1, +2, +3, +4, and +5 Da.

Micro scale chromatography of human plasma proteins for nano LC-ESI-MS/MS.

Organic precipitation of proteins with acetonitrile demonstrated complete protein recovery and improved chromatography of human plasma proteins. The separation of 25 μL of human plasma into 22 fractions on a QA SAX resin facilitated more effective protein discovery despite the limited sample size. Micro chromatography of plasma proteins over quaternary amine (QA) strong anion exchange (SAX) resins performed best, followed by diethylaminoethyl (DEAE), heparin (HEP), carboxymethyl cellulose (CMC), and propyl sulfate (PS) resins.