Improving limits of detection for B-type natriuretic peptide using PC-IDMS: an application of the ALiPHAT strategy.
Hydrophobic tagging of biomolecules has been reported by our group and others to increase their ionization efficiency during electrospray ionization and facilitate their detection by mass spectrometry. As such, hydrophobic tagging should provide a viable method for augmenting MS-based quantification of low abundance proteins by decreasing their detection limits. Herein we have evaluated two commercial alkylation reagents and several newly synthesized hydrophobic alkylation reagents for their utility in quantifying B-type Natriuretic Peptide, a low abundance cardiac biomarker, by protein cleavage isotope dilution mass spectrometry. For the cysteine containing tryptic peptide evaluated, a approximately 3.5-fold decrease in the detection limit was observed for the best performing hydrophobic reagent, 2-iodo-N-octylacetamide, relative to the commonly used alkylation reagent, iodoacetamide. Additionally, we have evaluated the use of nonpolar surface areas as a metric for assessing the effectiveness of the alkylation reagents in improving ESI response.
Shuford
Christopher M
CM
WM Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, USA.
Comins
Daniel L
DL
Whitten
Jerry L
JL
Burnett
John C
JC
Muddiman
David C
DC
D000477:Alkylating Agents / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D007460:Iodoacetamide / Q000737:chemistry; D007553:Isotope Labeling; D057230:Limit of Detection; D020097:Natriuretic Peptide, Brain / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D000477:Alkylating Agents; D020097:Natriuretic Peptide, Brain; D014357:Trypsin; D007460:Iodoacetamide