An immunoaffinity liquid chromatography–tandem mass spectrometry assay for the quantitation of matrix metalloproteinase 9 in mouse serum

Ocaña, Mireia Fernández; Neubert, Hendrik

doi:10.1016/j.ab.2010.01.002

Cited by 66 publications

(32 citation statements)

References 37 publications

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“…Thus, without the current detection threshold of probe 2, detection of active MMP-12 in BALf samples would have failed. It should also be noted that, by contrast to other reports dealing with the detection of MMPs, our approach involved no prefractionation or sample concentration (44,45). These prior steps may lead to a loss of low-abundance active forms of MMP.…”

Section: Discussionmentioning

confidence: 87%

Detection of Endogenous Matrix Metalloprotease-12 Active Form with a Novel Broad Spectrum Activity-based Probe*

Nury

Bregant

Czarny

et al. 2013

Journal of Biological Chemistry

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Background:The detection of MMP active forms remains a challenge. Results: An endogenous active form of MMP-12 was detected in animal fluids with a new activity-based probe. Conclusion:The presence of MMP active forms can be demonstrated only with a highly sensitive probe. Significance: It should be possible to validate active forms of MMP as potential biomarkers in different physiopathological contexts.

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Section: Discussionmentioning

confidence: 87%

Detection of Endogenous Matrix Metalloprotease-12 Active Form with a Novel Broad Spectrum Activity-based Probe*

Nury

Bregant

Czarny

et al. 2013

Journal of Biological Chemistry

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“…Achievable raw mass spectrometric detection limits are comparable to fused silicabased nanospray emitters (23 ), and we typically observe unchanged performance without any intervention often beyond 2 weeks of unattended and continuous operation. We have successfully used this emitter in other quantitative investigations (13,24 ).…”

Section: Liquid Chromatography Configurationmentioning

confidence: 99%

“…Although the original SISCAPA work relied on online nanoliter immunoaffinity columns based on Poros supports linked to nanoflow LC and nanospray mass spectrometry (6 ), off-line magnetic bead-based enrichment of enzymatically released peptides followed by either nano-or capillary LC-MS/MS has become a popular approach (7)(8)(9)(10)(11)(12). Bead-based sample preparation has the advantage that the antibody capture step can be done in parallel for multiple samples, and this process can be automated on a magnetic particle processor (11 ) or liquid-handling robotics, as shown for immunoaffinity enrichment of proteins (13,14 ). An extension of the magnetic bead SISCAPA work is the use of an inline bead-trap device to allow the quantification of lower-abundance peptides in a more automated fashion and with reduced off-line handling (15 ).…”

mentioning

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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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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“…When no protein or peptide purification step is employed, LC/MS sensitivity is significantly compromised due to matrix interference from the peptide-rich digest [24]. For high-sensitivity LC/MS applications, immunopurification is the most effective way to improve assay sensitivity and robustness by isolating target protein from matrix [25,26]. Further immunopurification of surrogate peptide from the digest could be carried out using an anti-peptide Ab [27].…”

Section: Resultsmentioning

confidence: 99%

Detection of cynomolgus monkey anti-protein XYZ antibody using immunocapture-LC/MS

Roos¹,

Chen²,

Vesapogu³

et al. 2016

J Appl Bioanal

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Although enzyme-linked immunosorbent assays and electrochemiluminescence (ECL) immunoassays are the most widely used platform for ADA detection, they may be compromised by drug interference. We describe here an alternate, free of drug interference, immunocapture-LC/MS methodology for detecting anti-protein XYZ antibody in cynomolgus monkey plasma. In our approach, ADA-protein XYZ complexes are captured by a mouse monoclonal anti-drug antibody on streptavidin magnetic beads and separated from monkey plasma by a magnet. After elution, ADA are digested with trypsin and detected by LC/MS using a surrogate peptide common to monkey IgG subclasses 1-4. The immunocapture-LC/MS assay was applied to support a toxicology study and results were in close agreement with those from a modified ECL immunoassay. To our knowledge, this is the first application of using LC/MS for monkey ADA detection.

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An immunoaffinity liquid chromatography–tandem mass spectrometry assay for the quantitation of matrix metalloproteinase 9 in mouse serum

Cited by 66 publications

References 37 publications

Detection of Endogenous Matrix Metalloprotease-12 Active Form with a Novel Broad Spectrum Activity-based Probe*

Detection of Endogenous Matrix Metalloprotease-12 Active Form with a Novel Broad Spectrum Activity-based Probe*

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

Detection of cynomolgus monkey anti-protein XYZ antibody using immunocapture-LC/MS

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