Online immunoaffinity liquid chromatography/tandem mass spectrometry determination of a type II collagen peptide biomarker in rat urine: Investigation of the impact of collision-induced dissociation fluctuation on peptide quantitation

Berna, Michael J.; Schmalz, C.; Duffin, Kevin L.; Mitchell, Peter G.; Chambers, M.G.; Ackermann, Brad

doi:10.1016/j.ab.2006.05.018

Cited by 51 publications

(33 citation statements)

References 10 publications

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“…We built immunoaffinity columns in-house according to the manufacturer's instructions and akin to procedures described for the same capture phase (18 ) or for similar capture phases (16,17 ). A detailed procedure can be found in online Supplemental Data 2.…”

Section: Methodsmentioning

confidence: 99%

“…The samples were heated to 100°C for 25 min and the plate was then allowed to cool to room temperature before adding 80 L of 68% acetonitrile together with 62.5 L of 2 ng/L Asp-N in 10 mmol/L Tris-HCl, pH 7.5. The samples were incubated for 16 We investigated dilution linearity using a 744 pmol/L (30 g/L) pepsinogen spike in saliva. Triplicate samples after 2-, 5-, and 10-fold dilution with 20 mmol/L ammonium bicarbonate, pH 7.9, were analyzed alongside the corresponding undiluted preparation.…”

Section: Assay Validationmentioning

confidence: 99%

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Online High-Flow Peptide Immunoaffinity Enrichment and Nanoflow LC-MS/MS: Assay Development for Total Salivary Pepsin/Pepsinogen

2010

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BACKGROUND Detection limit challenges associated with measuring low-abundance protein biomarkers can be addressed with hybrid immunoaffinity–mass spectrometric assays, such as antipeptide antibody capture followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Popular assay formats use magnetic bead–based immunoaffinity enrichment and nanoflow LC-MS/MS or high-flow immunoaffinity chromatography coupled online to conventional LC-MS/MS. As a proof of principle, we describe a novel online immunoaffinity LC-MS/MS configuration that combines high-flow peptide immunoaffinity enrichment and nanoflow LC-MS/MS. METHODS We configured and validated an assay for the measurement of total pepsin/pepsinogen from human saliva that uses a pepsinogen standard. Saliva was heat-inactivated to quench residual enzymatic activity and then digested with endoproteinase AspN. Online immunoaffinity enrichment using an antipeptide antibody directed against the pepsin C-terminal sequence, DRANNQVGLAPVA, was linked to nanoflow liquid chromatography and selected reaction monitoring mass spectrometry. We used the assay to measure pepsin/pepsinogen concentrations in human saliva from presumed healthy volunteers. RESULTS Heat inactivation at 100 °C for 25 min stabilized the target peptide. The final assay had <15% interassay relative error and <15% interassay CV across a range of 4.08–2980 pmol/L human pepsinogen (0.165–120 μg/L). Low but quantifiable signals were observed in some samples from presumed normal healthy volunteers ranging from 4.3 to 16.6 pmol/L (0.17–0.67 μg/L) total salivary pepsin/pepsinogen. CONCLUSIONS This assay approach provides a high-sensitivity platform for protein bioanalysis in the low picomolar range. It bears the potential to deliver additional data on the salivary occurrence of pepsin/pepsinogen with greater confidence than previously.

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Section: Methodsmentioning

confidence: 99%

Section: Assay Validationmentioning

confidence: 99%

Online High-Flow Peptide Immunoaffinity Enrichment and Nanoflow LC-MS/MS: Assay Development for Total Salivary Pepsin/Pepsinogen

2010

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“…Although selected reaction monitoring (SRM-MS) has been applied to the quantitation of plasma protein-derived peptides [15][16][17][18][19][20][21], a major limitation is that, due to ion suppression, the majority of biomarker proteins (e.g. PSA, CEA, and AFP present at ng/mL) cannot be detected in plasma in a mass spectrometry experiment without enrichment relative to large quantities of interfering proteins (e.g.…”

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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“…However, these signature peptide-specific antibodies may not be commercially available and require inhouse development increasing method development cost and time. Some methods have employed this type of immunocapture online using specialized columns containing analyte-specific antibodies [29,[36][37][38]. This workflow is extensively used for multiplexed biomarker assays [24,39].…”

Section: Immunocatpture Using Protein-specific Antibodiesmentioning

confidence: 99%

Internal Standards for Absolute Quantification of Large Molecules (Proteins) from Biological Matrices by LC-MS/MS

Faria¹,

Halquist²

2018

Calibration and Validation of Analytical Methods - A Sampling of Current Approaches

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Internal standardization plays a critical role in the performance of a bioanalytical method. There has been a tremendous increase in the popularity of using liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for quantitative bioanalysis of protein molecules. Protein, being too large to be directly analyzed by LC-MS/MS, is proteolyzed and a characteristic peptide is used as a surrogate analyte for quantification. Internal standardization in small molecules' analysis is straightforward, i.e., either a stable labeled isotope (SIL) form of the analyte or a structural analogue is used. As protein quantification involves protein digestion to yield peptides, there are more options for internal standardization. Currently, internal standard selection is based on the availability of the internal standards and the sample preparation workflow. A SIL-form of the analyte protein is the ideal internal standard. However, its use is limited due to cost and commercial availability. Alternatively, a SIL form the surrogate peptide analyte or a cleavable SIL-peptide can be used as an IS. For preclinical bioanalysis of humanized IgG antibody-based drugs, a universal SIL analogue protein has been effectively used as an internal standard. This chapter focuses on internal standardization for the quantitative analysis of proteins, such as biotherapeutics and biomarkers, using LC-MS/MS.

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Online immunoaffinity liquid chromatography/tandem mass spectrometry determination of a type II collagen peptide biomarker in rat urine: Investigation of the impact of collision-induced dissociation fluctuation on peptide quantitation

Cited by 51 publications

References 10 publications

Online High-Flow Peptide Immunoaffinity Enrichment and Nanoflow LC-MS/MS: Assay Development for Total Salivary Pepsin/Pepsinogen

Online High-Flow Peptide Immunoaffinity Enrichment and Nanoflow LC-MS/MS: Assay Development for Total Salivary Pepsin/Pepsinogen

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

Internal Standards for Absolute Quantification of Large Molecules (Proteins) from Biological Matrices by LC-MS/MS

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