Isotope dilution--mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I.
An enzymatic hydrolysis isotope dilution-mass spectrometric method was developed for reference quantification of specific proteins. The analytical procedure involved measuring a reproducibly hydrolyzed peptide (serving as the primary standard) unique to a specific protein. This new mass spectrometric method was evaluated by assessing the concentration of apolipoprotein (apo) A-I in the European Community Bureau of Reference (BCR) lyophilized Certified Reference Material (CRM 393). We used the method to make 96 measurements (4 replicate analyses of 4 enzymatic digests of 6 vials of BCR-CRM 393), which gave an average total protein mass of 1.048 mg (+/- 1.0% at 99% confidence limits). The total overall analytical CV was 3.95%. The results of this evaluation of our model approach to determine the concentration of a specific protein in a purified preparation demonstrated that our new mass spectrometric method can be used to measure apolipoproteins and other specific proteins without the use of epitopic immunoassay methods.
Barr
J R
JR
Division of Environmental Health Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Public Health Service, US Department of Health and Human Services, Atlanta, GA 30341-3724, USA.
Maggio
V L
VL
Patterson
D G
DG
Cooper
G R
GR
Henderson
L O
LO
Turner
W E
WE
Smith
S J
SJ
Hannon
W H
WH
Needham
L L
LL
Sampson
E J
EJ
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007201:Indicator Dilution Techniques; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010449:Peptide Mapping; D016339:Spectrometry, Mass, Fast Atom Bombardment; D014357:Trypsin / Q000378:metabolism
D016632:Apolipoprotein A-I; D010446:Peptide Fragments; D014357:Trypsin