scholarly journals PhosphoShield: Improving Trypsin Digestion of Phosphoproteins by Shielding the Negatively Charged Phosphate Moiety

2020 ◽  
Vol 31 (10) ◽  
pp. 2053-2060
Author(s):  
Julia A. Bubis ◽  
Vladimir Gorshkov ◽  
Mikhail V. Gorshkov ◽  
Frank Kjeldsen
Keyword(s):  
Trypsin Digestion ◽  
Phosphate Moiety

scholarly journals Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry

2021 ◽  
Vol 22 (6) ◽  
pp. 3166
Author(s):  
Jwala Priyadarsini Sivaccumar ◽  
Antonio Leonardi ◽  
Emanuela Iaccarino ◽  
Giusy Corvino ◽  
Luca Sanguigno ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Western Blotting ◽  
Cancer Biomarkers ◽  
Trypsin Digestion ◽  
Protein Fragments ◽  
Target Epitope ◽  
Potential Activity ◽  
Antibody Epitopes

Background: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb’s epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. Methods: We generated a mAb against PRAME immunizing mice with PRAME fragment 161–415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. Results: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202–212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. Conclusions: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody’s epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes.

scholarly journals Monoclonal antibody studies of creatine kinase. The ART epitope: evidence for an intermediate in protein folding

1989 ◽  
Vol 257 (2) ◽  
pp. 461-469 ◽  
Author(s):  
G E Morris
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Creatine Kinase ◽  
Specific Binding ◽  
Trypsin Digestion ◽  
Proteinase K ◽  
Enzyme Linked Immunosorbent Assay ◽  
Muscle Creatine Kinase ◽  
Western Blots ◽  
Methionine Residues

Chemical cleavage at cysteine residues with nitrothiocyanobenzoic acid shows that the last 98 amino acids of the 380-amino-acid sequence of chick muscle creatine kinase are sufficient for binding of the monoclonal antibody CK-ART. Removal of the last 30 amino acids by cleavage at methionine residues with CNBr results in loss of CK-ART binding. CK-ART binding is also lost when these C-terminal methionine residues are oxidized to sulphoxide, but binding is regained on reduction. Proteinase K ‘nicks’ native CK at a single site near the C-terminus and two fragments of 327 amino acides and 53 amino acids can be separated by subsequent SDS or urea treatment. CK-ART still binds normally to ‘nicked’ CK, which is enzymically inactive. After treatment with either urea (in a competition enzyme-linked immunosorbent assay) or SDS (on Western blots), however, CK-ART binds to neither of the two fragments, although these treatments do not affect binding to intact CK. This suggests that parts of both CK fragments contribute to the CK-ART epitope. CK-ART is both species- and isoenzyme-specific, binding only to chick M-CK. The only C-terminal regions containing chick-specific sequences are residues 300-312 and residues 368-371, the latter group being close to the essential methionine residues. We suggest that one, or possibly both, of these regions is involved in forming the conformational epitope on the surface of the CK molecule which CK-ART recognizes. Native CK is resistant to trypsin digestion. The C-terminal half of urea-treated and partly-refolded CK is also resistant to trypsin digestion, whereas the N-terminal half is readily digested. The results suggest a C-terminal region which can refold more rapidly than the rest of the CK molecule and provide evidence for an intermediate in CK refolding.

scholarly journals Isolation of peptides with immunoreactivity from OV albumin by trypsin digestion

FEBS Letters ◽  
1982 ◽  
Vol 141 (2) ◽  
pp. 225-228 ◽  
Author(s):  
Yoshihiro Tokota ◽  
Ayoko Matsushima ◽  
Yuji Inada
Keyword(s):  
Trypsin Digestion

Trypsin digestion of canine cardiac myosin

1976 ◽  
Vol 174 (1) ◽  
pp. 27-35 ◽  
Author(s):  
Ashok K. Bhan ◽  
Ashwani Malhotra
Keyword(s):  
Trypsin Digestion ◽  
Cardiac Myosin
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