Methods for on-chip protein analysis

Caputo, Emilia; Moharram, Ramy; Martin, Brian M.

doi:10.1016/s0003-2697(03)00361-0

Cited by 78 publications

(51 citation statements)

References 21 publications

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“…These substances can interfere with MS analysis and reduce assay sensitivity. 25 Results show that the enzymatic reactions produce the predicted Ang I and Ang II peptides with inhibition by specific inhibitors. For example, initial studies of ACE1 revealed that incubation of Ang I with plasma resulted in the formation of Ang II with a linear relationship between substrate depletion and product formation.…”

Section: Discussionmentioning

confidence: 97%

“…25,36 It provides a tool for the determination of biomarkers of physiological/pathological states. 25,36 Recently we used this emerging technology for the evaluation of ACE1 and renin activity in mice, using plasma sample sizes of Ͻ1 L. 24,37 The method does not require fluorogenic substrates, radioactivity, or laborious purifications steps. The objective of the present study was to determine whether this technology could be extended to the measurement of ACE2 activity.…”

Section: Discussionmentioning

confidence: 99%

“…The key issue behind the methodology is the ability to distinguish and quantify small molecular weight peptides. 25,26 This means that it can be applied to peptide sequencing, as well as quantification of enzymatic reactions. This is different from spectroscopic assays in which the index of activity is the change in color or fluorescence with no information regarding the identity of the specific peptides formed in the proteolytic reactions.…”

mentioning

confidence: 99%

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New Mass Spectrometric Assay for Angiotensin-Converting Enzyme 2 Activity

et al. 2006

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Abstract-A novel assay was developed for evaluation of mouse angiotensin-converting enzyme (ACE) 2 and recombinant human ACE2 (rACE2) activity. Using surface-enhanced laser desorption/ionization time of flight mass spectrometry (MS) with ProteinChip Array technology, ACE1 and ACE2 activity could be measured using natural peptide substrates.Plasma from C57BL/6 mice, kidney from wild-type and ACE2 knockout mice, and rACE2 were used for assay validation. Plasma or tissue extracts were incubated with angiotensin I (Ang I; 1296 m/z) or angiotensin II (Ang II; 1045 m/z). Reaction mixtures were spotted onto the ProteinChips WCX2 and peptides detected using surface-enhanced laser desorption/ionization time of flight MS. MS peaks for the substrates, Ang I and Ang II, and the generated peptides, Ang (1-7) and Ang (1-9), were monitored. The ACE2 inhibitor MLN 4760 (0.01 to 100 mol/L) significantly inhibited rACE2 activity (IC 50 ϭ3 nmol/L). Ang II was preferably cleaved by rACE2 (kmϭ5 mol/L), whereas Ang I was not a good substrate for rACE2. There was no detectable ACE2 activity in plasma. Assay specificity was validated in a model of ACE2 gene deletion. In kidney extract from ACE2-deficient mice, there was no generation of Ang (1-7) from Ang II. However, Ang (1-7) was produced when Ang I was used as a substrate. In conclusion, we developed a specific and sensitive assay for ACE2 activity, which used the natural endogenous peptide substrate Ang II. This approach allows for the rapid screening for ACE2, which has applications in drug testing, high-throughput enzymatic assays, and identification of novel substrates/inhibitors of the renin-angiotensin system. Key Words: angiotensin Ⅲ renin-angiotensin system Ⅲ mice Ⅲ angiotensin converting enzyme T he renin-angiotensin system (RAS) is a key regulator of cardiovascular and renal function, both under physiological and pathological conditions. 1

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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New Mass Spectrometric Assay for Angiotensin-Converting Enzyme 2 Activity

et al. 2006

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“…However, substantial caveats remain, including limited biomarker discovery in complex biological samples, PTM analysis of proteins altered with diseases, identification of proteins/peptides, and precise protein/ peptide quantitation (47). Platforms for direct on-chip sequencing of detected biomarkers by Q-TOF MS have become available, thus increasing the feasibility of the identification of discriminative proteins (48). SELDI-TOF MS has been applied to cancer tissue (49), plasma (50), serum (51), and nipple aspirate fluid (52).…”

Section: Proteomics Profiling Using Surface-enhanced Laser Desorptionmentioning

confidence: 99%

A Proteomic Primer for the Clinician

Guo

Fu²,

Eyk³

2007

Proceedings of the American Thoracic Society

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Proteomics is a rapidly developing field and it opens new horizons in many research areas of life sciences. In the field of medicine, proteomics promises to accelerate the discovery of new drug targets and protein disease markers useful for in vitro diagnosis. In this article, we review the current proteomics technologies for biomarker discovery and validation, which include two-dimensional gel electrophoresis, one-and two-dimensional liquid chromatography, and proteomic microarrays. We will also review proteomic strategies for protein-protein interactions and identification of posttranslational modifications. Selection of the more effective technology or combination of technologies is required to maximize the interpretation and utility of the data.

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“…En effet, elle utilise des substrats, dans des conditions non dénaturantes, dont la diversité des cibles auto-antigéniques n'est pas limitée par la technologie elle-même. Sa principale limite réside dans les difficultés d'identification et de caractérisation structurale des auto-antigènes reconnus [16] : les autoantigènes ne sont en effet pas ordonnés avant l'étape de chromatographie d'affinité, et leur identification nécessite, dans l'état actuel de la technologie, de mettre en oeuvre un travail d'analyse supplémentaire complexe. Par ailleurs, il est important de signaler que des systèmes de couplage de la chromatographie d'affinité à la spectrométrie de masse Maldi-Tof (matrix assisted laser desorption ionization) commencent à apparaître.…”

Section: Technologie Seldi (Surface-enhanced Laser Desorption/ionisatunclassified