Mass spectrometric characterization of limited proteolysis activity in human plasma samples under mild acidic conditions.
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.
Proteome Center Rostock, University Medicine Rostock, Rostock, Germany.
Jingzhi
J
Yang
Proteome Center Rostock, University Medicine Rostock, Rostock, Germany.
Claudia
C
Röwer
Proteome Center Rostock, University Medicine Rostock, Rostock, Germany.
Cornelia
C
Koy
Proteome Center Rostock, University Medicine Rostock, Rostock, Germany.
Manuela
M
Ruß
Analytical Chemistry Department, Institute of Chemistry, University of Rostock, Rostock, Germany; Cooperation Group of Comprehensive Molecular Analytics, Helmholtz Zentrum München, Munich, Germany.
Christopher P
CP
Rüger
Analytical Chemistry Department, Institute of Chemistry, University of Rostock, Rostock, Germany; Cooperation Group of Comprehensive Molecular Analytics, Helmholtz Zentrum München, Munich, Germany.
Ralf
R
Zimmermann
Division of Plastic Surgery and Hand Surgery, HELIOS Clinic Emil von Behring, Berlin, Germany.
Uwe
U
von Fritschen
Department of Cranio-Maxillofacial and Oral Surgery, University Hospital of Zürich, Zürich, Switzerland.
Marius
M
Bredell
Division of Plastic Surgery and Hand Surgery, HELIOS Clinic Emil von Behring, Berlin, Germany.
Juliane C
JC
Finke
Proteome Center Rostock, University Medicine Rostock, Rostock, Germany. Electronic address: michael.glocker@med.uni-rostock.de.
Michael O
MO
Glocker
D000136:Acid-Base Equilibrium; D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010949:Plasma / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D059748:Proteolysis; D020539:Sequence Analysis, Protein; D012709:Serum Albumin / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001798:Blood Proteins; D012709:Serum Albumin
Limited proteolysis; MALDI ToF MS; Plasma proteins; Protein structure; Tissue quality assay