scholarly journals Use of MALDI-TOF Mass Spectrometry for Specificity Studies of Biomedically Important Proteases

2002 ◽  
Vol 16 (3-4) ◽  
pp. 161-170 ◽  
Author(s):  
Jüri Siigur ◽  
Katrin Trummal ◽  
Külli TÕnismägi ◽  
Mari Samel ◽  
Ene Siigur ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Snake Venom ◽  
Serine Proteases ◽  
Factor Ix ◽  
Diagnostic Tools ◽  
Factor V ◽  
Factor X ◽  
Maldi Tof Mass Spectrometry ◽  
Maldi Tof ◽  
Vipera Lebetina

Proteases play crucial role starting from fertilization until to cell death. Our studies of the two Viperidae venoms (Levantine viperVipera lebetina, Common viperVipera berus) have demonstrated the existence of biomedically important proteases, both coagulants and anticoagulants that may be useful as diagnostic tools or potential therapeutics. We showed that venoms of both snakes contain: (i) metalloproteases and serine proteases that degrade fibrinogen, but not fibrin; (ii) factor X activators (VLFXA, VBFXAE); (iii) bradykinin-releasing serine proteases. AdditionallyVipera lebetinasnake venom contains thrombolytic fibrin degrading metalloenzyme (lebetase), HUVEC cell apoptosis inducing metalloprotease (VLAIP), factor V activator (VLFVA), thermostable β-fibrinogenase and α-fibrinogenase which has no homolog among known serine proteases. We examined the activity of snake venom proteases against bradykinin, substance P, insulin B-chain and 6–10 amino acid residues containing peptides synthesized according to potential cleavage regions of fibrinogen, factor X, factor IX, factor V, α2-macroglobulin bait region and pregnancy zone protein (PZP). We used MALDI TOF mass spectrometry technique for the discovery and identification of peptides released by protease hydrolysis. The sensitive and quick MALDI-TOF mass spectrometry methodology allows us to obtain the primary information about the substrate specificity of different proteases against various peptides and proteins.

scholarly journals Proteomic profiling of a snake venom using high mass detection MALDI-TOF mass spectrometry

2007 ◽  
Vol 18 (4) ◽  
pp. 600-606 ◽  
Author(s):  
Oscar Yanes ◽  
Francesc X. Avilés ◽  
Ryan Wenzel ◽  
Alexis Nazabal ◽  
Renato Zenobi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Snake Venom ◽  
High Mass ◽  
Proteomic Profiling ◽  
Mass Detection ◽  
Maldi Tof Mass Spectrometry ◽  
Maldi Tof

Molecular evolution of the vertebrate blood coagulation network

2003 ◽  
Vol 89 (03) ◽  
pp. 420-428 ◽  
Author(s):  
Colin Davidson ◽  
Robert Hirt ◽  
Kalpana Lal ◽  
Philip Snell ◽  
Greg Elgar ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Serine Proteases ◽  
Large Scale ◽  
Factor Ix ◽  
Supplementary Information ◽  
Factor Vii ◽  
Factor V ◽  
Factor X ◽  
Sequence Alignments ◽  
Comparative Sequence

SummaryIn mammalian blood coagulation 5 proteases, factor VII (FVII), factor IX (FIX), factor X (FX), protein C (PC) and prothrombin act with two cofactors factor V and factor VIII to control the generation of fibrin. Biochemical evidence and molecular cloning data have previously indicated that blood coagulation involving tissue factor, prothrombin and fibrinogen is present in all vertebrates. Using degenerate RT-PCR we have isolated and characterized novel cDNAs with sequence identity to the blood coagulation serine proteases and cofactors from chicken and the puffer fish (Fugu rubripes). Sequence alignments, phylogenetic and comparative sequence analysis all support the existence of the Gla-EGF1-EGF2-SP domain serine proteases FVII, FIX, FX, PC and the A1-A2-B-A3-C1-C2 domain protein cofactors FV and FVIII in these species. These results strongly suggest that the blood coagulation network is present in all jawed vertebrates and evolved before the divergence of tetrapods and teleosts over 430 million years ago; and that vertebrate blood coagulation may have benefited from two rounds of gene or whole genome duplication. Sequences identified in Fugu coding for additional FVII-like, FIX-like and PC-like sequences support the possibility of further tandem and large-scale duplications in teleosts. Comparative sequence analyses of amino acid residues in the active site region suggest these additional sequences have evolved new and as yet unknown functions.Supplementary information to this article available at both http://europium.csc.mrc.ac.uk and www.thrombosis-online.com

Behavior of serine proteases thrombin, trypsin, and chymotrypsin under conditions of MALDI-TOF-Mass spectrometry

2008 ◽  
Vol 44 (2) ◽  
pp. 75-79
Author(s):  
A. A. Poyarkov ◽  
T. Yu. Gromovoi ◽  
V. A. Pokrovskii ◽  
S. A. Poyarkova ◽  
V. P. Kukhar’
Keyword(s):  
Mass Spectrometry ◽  
Serine Proteases ◽  
Maldi Tof Mass Spectrometry ◽  
Maldi Tof

Spectrophotometric Determination of Factor Xa Generation in Factor IX Concentrates

1977 ◽  
Vol 37 (03) ◽  
pp. 535-540 ◽  
Author(s):  
D. S Pepper ◽  
D Banhegyi ◽  
Ann Howie
Keyword(s):  
Intrinsic Factor ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor V ◽  
Factor X ◽  
Generation Times ◽  
Incubation Periods ◽  
Multiple Sample ◽  
Recording Spectrophotometer

SummaryPrevious work from this department, concerned with testing the potential thrombogenicity of therapeutic factor IX concentrates, demonstrated that following recalcification of factor IX concentrates thrombin was generated within 3-30 minutes of incubation (Sas et al. 1975). The test developed (known as the TGt 50 test) is a two-stage assay and was thus found to be time consuming, tedious and tended to become inaccurate with long incubation periods and a large number of samples. A semiautomatic version of the test is reported in which the synthetic peptide Bz-ILE-GLU-GLY-ARG-pNA (S-2222) is added to recalcified, diluted factor IX concentrate in the micro-cuvette of a multiple sample recording spectrophotometer. Information can be obtained on (a) the amount of Xa (if any) present prior to recalcification (b) the initial amount of Xa formed and (c) the time taken to activate all factor X to Xa. Direct graphical interpretation shows a number of qualitative differences between commercial preparations, but by either of the criteria (b) or (c) above, it is possible to place the different products into “activated” and “non activated” groups such that both the Xa generation times and TGt 50 tests identify the same two groups of products. This agreement also indicates that the TGt 50 test is independent of the intrinsic factor V levels in the various concentrates.

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