title: Isotope dilution assay in peptide quantification: the challenge of microheterogeneity of internal standard.
abstract: Isotope dilution analysis allows quantitation of elements and different compounds in complex mixtures. The quantitation is based on a known amount of reference material (internal standard, IS) added to a sample that makes the result critically dependent on the value assigned to the standard. In the case of peptides, IS concentration is determined by nitrogen and amino acid analysis while purity is normally assessed by methods such as chromatography or electrophoresis that might not be able to detect many possible amino acid modifications, either naturally occurring or chemically induced. Microheterogeneity of the IS, if it is not accounted for when assigning a reference value to the standard, results in highly overestimated values in target analyte quantitation. In this viewpoint article, we illustrate the problem of internal standard microheterogeneity by analyzing synthetic human C-peptide labeled analogs.
authors: School of Medicine, University of Missouri, Columbia, MO, USA.
authors: Alexander V
authors: AV
authors: Stoyanov
authors: Eduard
authors: E
authors: Rogatsky
authors: Daniel
authors: D
authors: Stein
authors: Shawn
authors: S
authors: Connolly
authors: Curt L
authors: CL
authors: Rohlfing
authors: Randie R
authors: RR
authors: Little
mesh_terms: D000818:Animals; D006801:Humans; D007554:Isotopes / Q000032:analysis* / Q000737:chemistry; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012015:Reference Standards
chemical_list: D007554:Isotopes; D010455:Peptides
keywords: C-peptide; Isotope dilution assay; Microheterogeneity; Reference material
references: Rapid Commun Mass Spectrom. 1997;11(9):1015-24
references: 9204576
references: Proteomics. 2009 Mar;9(5):1124-7
references: 19253279
references: Proteomics Clin Appl. 2010 Jan;4(1):106-11
references: 21137020
references: J Biol Chem. 1997 May 9;272(19):12513-22
references: 9139702
references: J Chromatogr Sep Tech. 2013;4(3):
references: 31942247
references: Clin Chem. 2006 May;52(5):872-9
references: 16556683
references: Rapid Commun Mass Spectrom. 2005;19(23):3600-2
references: 16276487
references: Biol Mass Spectrom. 1991 Mar;20(3):130-8
references: 2069984
references: J Chromatogr A. 2011 Dec 23;1218(51):9244-9
references: 22098929
references: Mol Cell Proteomics. 2002 May;1(5):376-86
references: 12118079
references: J Chromatogr A. 2000 Oct 27;896(1-2):275-8
references: 11093662
references: J Sep Sci. 2006 Mar;29(4):529-37
references: 16583691
references: Curr Opin Biotechnol. 1993 Feb;4(1):14-9
references: 7763387
references: West Indian Med J. 2000 Jun;49(2):138-42
references: 10948853
references: Diabetologia. 2002 Nov;45(11):1523-7
references: 12436335
references: N Engl J Med. 1969 May 15;280(20):1106-13
references: 4305118