Document Detail


Coupling of a vented column with splitless nanoRPLC-ESI-MS for the improved separation and detection of brain natriuretic peptide-32 and its proteolytic peptides.
MedLine Citation:
PMID:  19269262     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The circulating concentration of a biomarker for congestive heart failure, Brain (B-type) Natriuretic Peptide (BNP-32), is measured using ELISA based assays in order to rapidly diagnose and monitor disease progression. The lack of molecular specificity afforded by these assays has recently come into question as emerging studies indicate there are potentially multiple heterogeneous forms of BNP in circulation with immunoreactive capabilities. In order to better understand the molecular biology of BNP-32 as it relates to congestive heart failure, it would thus be advantageous to use a detection platform such as Fourier transform ion cyclotron resonance mass spectrometry. This high resolving power mass spectrometer can provide unparalleled molecular specificity and can facilitate identification and characterization of the various molecular forms across all disease states. Unfortunately, BNP circulates at low concentrations (as low as 3fmol/mL). Thus, it will require a collaborative effort from a number of orthogonal front-end technologies to overcome the disconnect between the practical detection limits of this instrument platform and the physiological levels of BNP-32 and its alternative molecular forms. Herein, we begin optimization of these front-end techniques by first enhancing the conditions for online nanoLC-ESI-MS separations of BNP-32 and its proteolytic fragments. Through extensive analysis of various chromatographic parameters we determined that Michrom Magic C8 stationary phase used in conjunction with a continuous, vented column configuration provided advanced chromatographic performance for the nano-flow separations involving intact BNP-32 and its associated tryptic peptides. Furthermore, conditions for the tryptic digestion of BNP-32 were also studied. We demonstrate that the use of free cysteine as an alkylation quenching agent and a secondary digestion within the digestion scheme can provide targeted tryptic peptides with increased abundances. Combined, these data will serve to further augment the detection of BNP-32 by LC-MS.
Authors:
Genna L Andrews; Christopher M Shuford; John C Burnett; Adam M Hawkridge; David C Muddiman
Publication Detail:
Type:  Evaluation Studies; Journal Article; Research Support, N.I.H., Extramural     Date:  2009-02-24
Journal Detail:
Title:  Journal of chromatography. B, Analytical technologies in the biomedical and life sciences     Volume:  877     ISSN:  1873-376X     ISO Abbreviation:  J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.     Publication Date:  2009 Apr 
Date Detail:
Created Date:  2009-03-23     Completed Date:  2009-04-07     Revised Date:  2014-09-18    
Medline Journal Info:
Nlm Unique ID:  101139554     Medline TA:  J Chromatogr B Analyt Technol Biomed Life Sci     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  948-54     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Brain Chemistry*
Chromatography, Liquid / methods*
Molecular Sequence Data
Natriuretic Peptide, Brain / chemistry*,  isolation & purification,  pharmacokinetics*
Spectrometry, Mass, Electrospray Ionization / methods*
Grant Support
ID/Acronym/Agency:
5R01HL036634/HL/NHLBI NIH HHS; 5T32GM00-8776-08/GM/NIGMS NIH HHS; R01 HL036634/HL/NHLBI NIH HHS; R01 HL036634-21/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
114471-18-0/Natriuretic Peptide, Brain
Comments/Corrections

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