Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA)

Anderson, N. Leigh; Anderson, Norman G.; Haines, Lee R.; Hardie, Darryl B.; Olafson, Robert W.; Pearson, Terry W.

doi:10.1021/pr034086h

Cited by 761 publications

(651 citation statements)

References 25 publications

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“…23,24 However, these techniques are not suitable for the assignment of values to reference materials. A reference measurement procedure must be critically investigated as to all potential sources of error, which must be identified, evaluated, and mitigated to the greatest extent possible.…”

Section: Discussionmentioning

confidence: 99%

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

Kilpatrick

Bunk

2009

Anal. Chem.

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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...

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Section: Discussionmentioning

confidence: 99%

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

Kilpatrick

Bunk

2009

Anal. Chem.

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“…P01011) and tumor necrosis factor alpha (TNFα, accn. P01375) peptides were described previously [22]. Stable isotope standards of AAC (EIGELYLPK, 13 C-and 15 N-labeled amino acid in italics) and TNFα (DLSLISPLAQAVR) were obtained from Cell Signaling Technologies (Beverly, MA).…”

Section: Methodsmentioning

confidence: 99%

“…albumin 50 mg/mL, globulin 35 mg/mL). To this end, we previously described a technology, "Stable Isotope Standards with Capture by Anti-Peptide Antibodies" (SISCAPA) in which anti-peptide antibodies immobilized on 100 nanoliter affinity columns were used to enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence [22]. Upon elution from the anti-peptide antibody supports, electrospray ionization (ESI) MS was used to quantify the peptides (natural and labeled).…”

Section: Introductionmentioning

confidence: 99%

Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers

Whiteaker

Zhao

Zhang

et al. 2007

Analytical Biochemistry

256

208

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A major bottleneck for validation of new clinical diagnostics is the development of highly sensitive and specific assays for quantifying proteins. We previously described a method, Stable Isotope Standards with Capture by Anti-Peptide Antibodies, wherein a specific tryptic peptide is selected as a stoichiometric representative of the protein from which it is cleaved, is enriched from biological samples using immobilized antibodies, and is quantitated using mass spectrometry against a spiked internal standard to yield a measure of protein concentration. In this report, we optimize a magnetic bead-based platform amenable to high throughput peptide capture and demonstrate that antibody capture followed by mass spectrometry can achieve ion signal enhancements on the order of 10 3 , with precision (CVs <10%) and accuracy (relative error ~20%) sufficient for quantifying biomarkers in the physiologically relevant ng/mL range. These methods are generally applicable to any protein or biological fluid of interest and hold great potential for providing a desperately needed bridging technology between biomarker discovery and clinical application.

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“…The enriched sample is then introduced into a mass spectrometer for analysis. Both relative and absolute quantification information about the protein of interest is then gathered [49]. This method was further optimized for greater precision and accuracy by Whiteaker et al, reporting a physiologically relevant sensitivity in the ng/mL range [50].…”

Section: Additional Targeted Proteomics Methodsmentioning

confidence: 99%

Current affairs in quantitative targeted proteomics: multiple reaction monitoring-mass spectrometry

Yocum¹,

Chinnaiyan²

2009

Briefings in Functional Genomics and Proteomics

109

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Quantitative targeted proteomics has recently taken front stage in the proteomics community. Centered on multiple reaction monitoring^mass spectrometry (MRM^MS) methodologies, quantitative targeted proteomics is being used in the verification of global proteomics data, the discovery of lower abundance proteins, protein post-translational modifications, discrimination of select highly homologous protein isoforms and as the final step in biomarker discovery. An older methodology utilized with small molecule analysis, the proteomics community is making great technological strides to develop MRM^MS as the next method to address previously challenging issues in global proteomics experimentation, namely dynamic range, identification of post-translational modifications, sensitivity and selectivity of measurement which will undoubtedly further biomedical knowledge.This brief review will provide a general introduction of MRM^MS and highlight its novel application for targeted quantitative proteomic experimentations.

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Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA)

Cited by 761 publications

References 25 publications

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

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

Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers

Current affairs in quantitative targeted proteomics: multiple reaction monitoring-mass spectrometry

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