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Mass spectrometric characterization of limited proteolysis activity in human plasma samples under mild acidic conditions.
Methods 89, 30-37 (2015)
We developed a limited proteolysis assay for estimating dynamics in plasma-borne protease activities using MALDI ToF MS analysis as readout. A highly specific limited proteolysis activity was elicited in human plasma by shifting the pH to 6. Mass spectrometry showed that two singly charged ion signals at m/z 2753.44 and m/z 2937.56 significantly increased in abundance under mild acidic conditions as a function of incubation time. For proving that a provoked proteolytic activity in mild acidic solution caused the appearance of the observed peptides, control measurements were performed (i) with pepstatin as protease inhibitor, (ii) with heat-denatured samples, (iii) at pH 1.7, and (iv) at pH 7.5. Mass spectrometric fragmentation analysis showed that the observed peptides encompass the amino acid sequences 1-24 and 1-26 from the N-terminus of human serum albumin. Investigations on peptidase specificities suggest that the two best candidates for the observed serum albumin cleavages are cathepsin D and E. Reproducibility, robustness, and sensitivity prove the potential of the developed limited proteolysis assay to become of clinical importance for estimating dynamics of plasma-borne proteases with respect to associated pathophysiological tissue conditions.
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Publikationstyp
Artikel: Journalartikel
Dokumenttyp
Wissenschaftlicher Artikel
Schlagwörter
Limited Proteolysis ; Maldi Tof Ms ; Plasma Proteins ; Protein Structure ; Tissue Quality Assay
ISSN (print) / ISBN
1046-2023
e-ISSN
1095-9130
Zeitschrift
Methods
Quellenangaben
Band: 89,
Seiten: 30-37
Verlag
Elsevier
Verlagsort
Amsterdam [u.a.]
Nichtpatentliteratur
Publikationen
Begutachtungsstatus
Peer reviewed