C-Terminal sequencing of peptide hormones using carboxypeptidase Y and SELDI-TOF mass spectrometry

Cool, David R.; Hardiman, Atira

doi:10.2144/04361bm01

Cited by 14 publications

(11 citation statements)

References 12 publications

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“…1). These peaks have previously been identified as des-acetyl--melanocyte stimulating hormone (MSH), -MSH and di-acetyl--MSH (1620, 1660 and 1702 daltons respectively) (Cool & Hardiman 2004). In the second region, ion species between 2350 and 2506 daltons were also identified in the heterozygote and normal genotype, but were absent from the PC2-Null genotype (b in Fig.…”

Section: Proteomic Analysis Of Neurointermediate Lobe Pituitaries By mentioning

confidence: 83%

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Endocrinomic profile of neurointermediate lobe pituitary prohormone processing in PC1/3- and PC2-Null mice using SELDI-TOF mass spectrometry

Hardiman

Friedman²,

Grunwald³

et al. 2005

Journal of Molecular Endocrinology

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Pro-vasopressin and pro-oxytocin are prohormones processed in the neurointermediate lobe pituitary to form the biologically active peptide hormones, arginine vasopressin (AVP) and oxytocin. Neurointermediate lobe pituitaries from normal (+/+), heterozygous (+/−), PC2-Null (−/−), PC1/3-Null and oxytocin-Null mice were analyzed by SELDI-TOF mass spectroscopy for the peptide hormone products, AVP, oxytocin and neurophysin I and II. Molecular ion species with masses characteristic of oxytocin, AVP, neurophysin I and II, i.e. 1009·41, 1084·5, 9677 and 9679 daltons respectively, were identified in all but the oxytocin-Null mice by comparison with synthetic standards or by C-terminal sequence analysis. Other ion species were found specifically in PC2-Null, heterozygote or normal mice. The results indicate that, in mice, both PC1/3 or PC2 enzyme activity are capable, but not required to correctly process pro-vasopressin or pro-oxytocin to their constituent active peptide hormones.

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Section: Proteomic Analysis Of Neurointermediate Lobe Pituitaries By mentioning

confidence: 83%

“…Neurointermediate lobe tissue lysates (1 µg protein) were spotted on an H4 ProteinChip and air-dried, as previously described (Cool & Hardiman 2004). The ProteinChip was placed in a 'moist chamber' -a pipette tip box (with lid) containing a wet paper towel.…”

Section: On-chip C-terminal Peptide Sequencingmentioning

confidence: 99%

Endocrinomic profile of neurointermediate lobe pituitary prohormone processing in PC1/3- and PC2-Null mice using SELDI-TOF mass spectrometry

Hardiman

Friedman²,

Grunwald³

et al. 2005

Journal of Molecular Endocrinology

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“…The reaction mixture (1 L) was then spotted onto ProteinChip Arrays and analyzed on as previously described. 17 Briefly, after the incubation period, 1 L of the reaction mixtures were spotted onto ProteinChip WCX2 and incubated for an extra 15 minutes in a humidified chamber at 37°C. ProteinChip were washed with deionized water (5 L, 3 times) to remove nonbound proteins, salts, and other contaminants.…”

Section: Seldi-tof-msmentioning

confidence: 99%

“…It is particularly useful for quantification of low-molecular-weight peptides and has been used for on-chip enzymatic peptide sequencing. 16,17 However, it has not yet been applied to a great extent to measurement of enzyme activity.…”

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Novel Mass Spectrometric Methods for Evaluation of Plasma Angiotensin Converting Enzyme 1 and Renin Activity

2005

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Abstract-This article demonstrates the applicability of quantitative proteomics to assays of proteolytic enzyme activity.A novel assay was developed for measurement of renin and angiotensin-converting enzyme (ACE) activity in plasma.The method was validated in animal models associated with alterations of the renin angiotensin system (RAS). Using surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) with a ProteinChip Array technology, plasma renin and ACE1 could be measured in Ͻ0.5 L of plasma. Plasma is incubated with peptide substrates for renin and ACE, tetradecapeptide (TDP), and angiotensin I (Ang I), respectively. The reactions mixtures are spotted onto the ProteinChip WCX2 and detected using SELDI-TOF-MS. Peak height or area under curve for TDP, Ang I, and angiotensin II (Ang II) peaks are measured. There was a linear relationship between disappearance of substrate and appearance of products for both renin and ACE (R 2 ϭ0.95 to 0.98). ACE1 activity was blocked with chelating agents (EDTA and 1,10 phenanthrolene), indicating action of a metalloprotease. The ACE1 inhibitor, captopril, selectively blocked ACE1. Renin activity was specifically blocked with renin inhibitor and was not affected by phenanthrolene or captopril. Animal models tested were Ang AT1a receptor-deficient and streptozotocin (STZ) diabetic mice. Plasma renin activity was increased Ͼ2-fold in AT1aϪ/Ϫ as compared with AT1a ϩ/ϩ . In STZ diabetic mice, ACE1 was increased 2-fold as compared with controls. The advantage of the method is that it is tagless, does not require additional purification steps, and is extremely sensitive. The approach can be multiplexed and used for identification of novel substrates/inhibitors of the RAS.

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“…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.…”

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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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C-Terminal sequencing of peptide hormones using carboxypeptidase Y and SELDI-TOF mass spectrometry

Cited by 14 publications

References 12 publications

Endocrinomic profile of neurointermediate lobe pituitary prohormone processing in PC1/3- and PC2-Null mice using SELDI-TOF mass spectrometry

Endocrinomic profile of neurointermediate lobe pituitary prohormone processing in PC1/3- and PC2-Null mice using SELDI-TOF mass spectrometry

Novel Mass Spectrometric Methods for Evaluation of Plasma Angiotensin Converting Enzyme 1 and Renin Activity

New Mass Spectrometric Assay for Angiotensin-Converting Enzyme 2 Activity

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