title: Two new types of assays to determine protein concentrations in biological fluids using mass spectrometry of intact proteins with cystatin C in spinal fluid as an example.
abstract: There is no reference method that is generally acknowledged to be unbiased for the determination of the concentration of any protein in biological fluids. This is probably because mass spectrometry (MS) methods acknowledged as reference methods for determination of low molecular mass substances in biological fluids, e.g. creatinine, have been difficult to adapt for proteins. Here we suggest two tentative MS methods, which might be used as reference methods for the determination of protein concentrations in biological fluids. One is based upon the addition to the fluid of a non-proteome reference protein, very similar to the one to be measured, and analyzing the ratio between the corresponding peaks in a selected ion monitoring (SIM) chromatogram. We call this method LC-MS-NPRP (NPRP, Non-Proteome Reference Protein). The other method is based upon the classical standard addition assay for low molecular mass substances. The results of these assays for cystatin C in spinal fluid were compared to those obtained by an immunoassay. Both methods indicated lower concentration than the immunoassay. This was found to be due to the presence of a significant fraction of monohydroxylated cystatin C in spinal fluid. It turned out that the sum of the unhydroxylated and hydroxylated cystatin C concentrations, determined by either of the two MS methods, were close to the results obtained by the immunoassay. These MS-based methods analyze intact proteins and therefore seem more suitable for the determination of protein concentrations in biological fluids than other MS-based methods requiring proteolytic degradation with its inherent lack of precision.
authors.affiliation: Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry, Uppsala University , Uppsala , Sweden.
authors.forename: Niklas
authors.initials: N
authors.lastname: Tyrefors
authors.forename: Peter
authors.initials: P
authors.lastname: Michelsen
authors.forename: Anders
authors.initials: A
authors.lastname: Grubb
mesh_terms: D002853:Chromatography, Liquid; D055316:Cystatin C / Q000134:cerebrospinal fluid*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D055316:Cystatin C
keywords: Bone metabolism; immunoassays; kidney disease; microheterogeneity