Reference Measurement Procedure Development for C-Reactive Protein in Human Serum

Abstract

This paper describes the development of a reference measurement procedure to quantify human C-reactive protein (CRP) in serum using affinity techniques prior to tryptic digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the certification of reference materials in clinically relevant ranges. The absence of a suitable internal standard for the CRP measurement, necessary to eliminate potential measurement bias in both the affinity purification and trypsin digestion steps, was addressed using the method of standard addition. The standard addition quantification approach was combined with affinity purification, using an anti-CRP monoclonal antibody conjugated to polystyrene beads, trypsin digestion of the purified protein, and LC-MS/MS analysis of CRP tryptic peptides. The effectiveness of intact protein affinity purification was evaluated through the measurement of CRP in several serum-based CRP control materials, yielding levels that were comparable to their expected mean concentration values. Quantitative results were confirmed with an external calibration approach. This study demonstrates the feasibility of affinity purification with LC-MS/MS for the reference measurement procedure development of low abundance serum protein analytes.Reference measurement procedures are used for a variety of purposes including (1) the assessment of the measurement quality of routine measurement procedures, (2) the value assignment of high-order calibration solutions (the so-called "master calibrators") for routine assays, and (3) the value assignment of certified reference materials. 1,2 In these roles, reference measurement procedures are considered "higher-order" measurement procedures, as they aim to achieve a higher level of accuracy and precision than the routine assay for the analyte being measured, producing a value with a low uncertainty of defined magnitude. In order for a reference measurement procedure to be fit for this purpose, it must be thoroughly evaluated to identify sources of bias and assess their magnitude. [3][4][5] Minimizing or eliminating bias is necessary to measure the most accurate, true concentration of the analyte. Because of the need for high levels of accuracy and precision, reference measurement procedures are typically low-throughput, labor intensive, and often costly to perform, in sharp contrast to what is desirable for routine measurements.Reference measurement procedures are key elements in the establishment of measurement standardization and metrological traceability in clinical chemistry. For more than 30 years, isotope dilution mass spectrometry (IDMS) has been one of the analytical techniques used frequently in reference measurement procedures, particularly for small molecule and inorganic analytes of clinical importance. 6 Despite this, few reference measurement procedures exist for clinically relevant proteins. This is one reason why so many clinical protein analytes are measured in arbitrary International Units (IUs); without a reference measurement procedure to...

LC–MS/MS for protein and peptide quantification in clinical chemistry

“…For many immunoassays, there is an urgent need for reliable reference methodology to verify calibrators and assay performance [31,32,[64][65][66][67][68]. SRM assays could represent a reference measurement procedure which allows measurements of highest metrological quality.…”

Current Trends in Magnetic Particle Enrichment for Mass Spectrometry-Based Analysis of Cardiovascular Protein Biomarkers

“…Analysis by isotope dilution (ID)-MS is considered the gold standard for protein or peptide quantification and is the preferred method for clinical validation of protein biomarkers. Measurements by ID-MS are more accurate and precise because the method requires the use of an isotopically labeled internal standard analogous to the target protein to account for sample processing and instrument variability [34,40]. MS has the ability to distinguish the target protein from the isotopi- Table 1.…”

“…Diagnosis of AMI and risk stratification of heart disease [20][21][22][23][24][25][26][27][28] Myoglobin [29] Biomarkers of myocardial stress Brain natriuretic peptides Aid in the diagnosis of heart failure [30][31][32][33] N-terminal of the prohormone of BNP [32] Biomarkers of inflammation C-reactive protein Elevated levels correlate to cardiovascular risk [34][35][36][37] IL-6, IL-33 [20,32] AMI: Acute myocardial infarction; BNP: B-type natriuretic peptide.…”

“…Strategies incorporating immunocapture coupled with targeted MS are currently capable of quantifying proteins from biofluids with limits of quantification (LOQs) in the ng/ml range [20,38]. However, at normal levels, protein biomarkers of cardiac disease typically range from ≈0-5 pg/ml (cTn and IL-6) to 1000 ng/ml (CRP) in plasma [34,44,53]. CVD-related proteins in the lower concentration range are below the LODs for current MS instrumentation using targeted and nontargeted proteomic approaches [34,44,54].…”

Analysis of the Physicochemical State of C-Reactive Protein in Different Preparations Including 2 Certified Reference Materials

“…In the case where all CRP polypeptides are claimed to be measured, the presence of monomeric CRP, as a reference material artifact, will lead to a break in traceability. For example, LC-MS-based methods are being developed for CRP (21 ). These methods measure all the polypeptide chains, including those in the monomeric form.…”