Chromogranin A from Bovine Adrenal Medulla: Molecular Characterization of Glycosylations, Phosphorylations, and Sequence Heterogeneities by Mass Spectrometry

Chromogranin A (CGA) is a protein that is stored and released together with neurotransmitters and hormones in the nervous, endocrine and diffuse neuroendocrine systems. As human vasostatins I and II [CGA(1–76) and CGA(1–113), respectively] have been reported to affect vessel motility and exert concentration‐dependent cardiosuppressive effects on isolated whole heart preparations of eel, frog and rat (i.e. negative inotropism and antiadrenergic activity), we investigated the presence of vasostatin‐containing peptides in rat heart. Rat heart extracts were purified by RP‐HPLC, and the resulting fractions analyzed for the presence of CGA N‐terminal fragments using dot‐blot analysis. CGA‐immunoreactive fractions were submitted to western blot and MS analysis using the TOF/TOF technique. Four endogenous N‐terminal CGA‐derived peptides [CGA(4–113), CGA(1–124), CGA(1–135) and CGA(1–199)] containing the vasostatin sequence were characterized. The following post‐translational modifications of these fragments were identified: phosphorylation at Ser96, O‐glycosylation (trisaccharide, NAcGal‐Gal‐NeuAc) at Thr126, and oxidation at three methionine residues. This first identification of CGA‐derived peptides containing the vasostatin motif in rat heart supports their role in cardiac physiology by an autocrine/paracrine mechanism.

show abstract

“…Among these, Ser96 in rat CGA corresponds to a previously described phosphorylated residue (Ser81) in the bovine CGA sequence (Fig. 5A) [17,38].…”

Section: Resultsmentioning

confidence: 99%

“…Post‐translational modifications (phosphorylation and O‐glycosylation) of bovine and human CGA have been identified [14–17]. Phosphorylations are present all along the sequence, whereas glycosylation sites are mainly located in the median part of CGA.…”

mentioning

confidence: 99%

Characterization of natural vasostatin‐containing peptides in rat heart

Glattard

Angelone

Strub³

et al. 2006

The FEBS Journal

View full text Add to dashboard Cite

show abstract

“…7 for the above-mentioned standard proteins. The technique is very reliable and has been widely used to identify peptide candidates [28,120,121,122,123,124,125], which then were fragmented with SIM-LC-MS/MS [82]. Incomplete dephosphorylation is occasionally observed [43].…”

Section: Protein Cleavagementioning

confidence: 99%

The impact of chromatography and mass spectrometry on the analysis of protein phosphorylation sites

Zeller

König

2004

Analytical and Bioanalytical Chemistry

View full text Add to dashboard Cite

Protein phosphorylation analysis is an enormous challenge. This review summarises the currently used techniques, which are based on radiolabelling and mass spectrometry as well as electrophoretic and chromatographic separation. Many methods exist, but there is still no single procedure applicable to all phosphoproteins. MS is able to deliver information about the location of phosphorylation sites, but phosphospecific properties with respect to ionisation present obstacles. Therefore, multidimensional approaches involving several analytical methods are often necessary to conquer phosphorylation site identification.

show abstract

“…Indeed, if the PTM in bacteria are not too frequent, there is good evidence that most of the proteins in higher organisms are modified and sometimes in a very unexpected manner. For example, it has been found that some proteins could have up to five phosphorylation sites [21]. As already stated, 2-D gel electrophoresis is technically able to separate these different isoforms.…”

Section: Introductionmentioning

confidence: 96%

Protein fractionation in a multicompartment device using Off‐Gel™ isoelectric focusing

et al. 2003

View full text Add to dashboard Cite

A new protein fractionation technique based on off-gel isoelectric focusing (IEF) is presented, where the proteins are separated according to their isoelectric point (pI) in a multiwell device with the advantage to be directly recovered in solution for further analysis. The protein fractions obtained with this technique have then been characterized with polymer nanoelectrospray for mass spectrometry (MS) analyses or with Bioanalyzer for mass identification. This methodology shows the possibility of developing alternatives to the classical two-dimensional (2-D) gel electrophoresis. One species numerical simulation of the electric field distribution during off-gel separation is also presented in order to demonstrate the principle of the purification. Experiments with pI protein markers have been carried out in order to highlight the kinetics and the efficiency of the technique. Moreover, the resolution of the fractionation was shown to be 0.1 pH unit for the separation of beta-lactoglobulin A and B. In addition, the isoelectric fractionation of an Escherichia coli extract was performed in standard solubilization buffer to demonstrate the performances of the technique, notably for proteomics applications.

show abstract

Chromogranin A from Bovine Adrenal Medulla: Molecular Characterization of Glycosylations, Phosphorylations, and Sequence Heterogeneities by Mass Spectrometry

Cited by 10 publications

References 47 publications

Characterization of natural vasostatin‐containing peptides in rat heart

Characterization of natural vasostatin‐containing peptides in rat heart

The impact of chromatography and mass spectrometry on the analysis of protein phosphorylation sites

Protein fractionation in a multicompartment device using Off‐Gel™ isoelectric focusing

Contact Info

Product

Resources

About