scholarly journals Bovine Complex I Is a Complex of 45 Different Subunits

2006 ◽  
Vol 281 (43) ◽  
pp. 32724-32727 ◽  
Author(s):  
Joe Carroll ◽  
Ian M. Fearnley ◽  
J. Mark Skehel ◽  
Richard J. Shannon ◽  
Judy Hirst ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mass ◽  
Electrospray Ionization ◽  
Complex I ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Intact Protein ◽  
Ionization Mass ◽  
Peptide Bond Cleavage ◽  
Membrane Bound

Mammalian mitochondrial complex I is a multisubunit membrane-bound assembly with a molecular mass approaching 1 MDa. By comprehensive analyses of the bovine complex and its constituent subcomplexes, 45 different subunits have been characterized previously. The presence of a 46th subunit was suspected from the consistent detection of a molecular mass of 10,566 by electrospray ionization mass spectrometry of subunits fractionated by reverse-phase high pressure liquid chromatography. The component was found associated with both the intact complex and subcomplex Iβ, which represents most of the membrane arm of the complex, and it could not be resolved chromatographically from subunit SGDH (the subunit of bovine complex I with the N-terminal sequence Ser-Gly-Asp-His). It has now been characterized by tandem mass spectrometry of intact protein ions and shown to be a C-terminal fragment of subunit SGDH arising from a specific peptide bond cleavage between Ile-55 and Pro-56 during the electrospray ionization process. Thus, the subunit composition of bovine complex I has been established. It is a complex of 45 different proteins plus non-covalently bound FMN and eight iron-sulfur clusters.

scholarly journals Iodine induced alteration in immunological and biochemical properties of thyroglobulin.

1993 ◽  
Vol 40 (2) ◽  
pp. 237-240 ◽  
Author(s):  
A Gardas ◽  
H Domek
Keyword(s):  
Molecular Mass ◽  
Bond Cleavage ◽  
Coupling Reaction ◽  
Peptide Bond ◽  
Biochemical Properties ◽  
Disulphide Bridge ◽  
Peptide Bond Cleavage ◽  
Epitope Structure ◽  
Iodide Solution ◽  
Ph Dependent

The influence of iodine-iodide solution on the biochemical and immunological properties of human thyroglobulin (hTg) were studied. Human Tg preincubated with the iodine-iodide solution is split to small molecular mass fragments after disulphide bridge reduction with dithiothreitol. The peptide bond cleavage by iodine pretreatment and reduction is possibly linked with the coupling reaction of diiodotyrosyl residues. Pretreatment of hTg with iodine-iodide solution at 1-10 microM decreased the binding of autoantibodies to hTg. The iodine-iodide induced inactivation of hTg autoepitopes is pH dependent and is possibly caused by iodination of tyrosyl residues present in the epitope structure.

scholarly journals Evolution of a mass spectrometry-grade protease with PTM-directed specificity

2016 ◽  
Vol 113 (51) ◽  
pp. 14686-14691 ◽  
Author(s):  
Duc T. Tran ◽  
Valerie J. Cavett ◽  
Vuong Q. Dang ◽  
Héctor L. Torres ◽  
Brian M. Paegel
Keyword(s):  
Mass Spectrometry ◽  
Posttranslational Modifications ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Arginine Deiminase ◽  
Peptide Bond Cleavage ◽  
Mass Spectral ◽  
Site Mapping ◽  
Fluorogenic Probes ◽  
Site Identification

Mapping posttranslational modifications (PTMs), which diversely modulate biological functions, represents a significant analytical challenge. The centerpiece technology for PTM site identification, mass spectrometry (MS), requires proteolytic cleavage in the vicinity of a PTM to yield peptides for sequencing. This requirement catalyzed our efforts to evolve MS-grade mutant PTM-directed proteases. Citrulline, a PTM implicated in epigenetic and immunological function, made an ideal first target, because citrullination eliminates arginyl tryptic sites. Bead-displayed trypsin mutant genes were translated in droplets, the mutant proteases were challenged to cleave bead-bound fluorogenic probes of citrulline-dependent proteolysis, and the resultant beads (1.3 million) were screened. The most promising mutant efficiently catalyzed citrulline-dependent peptide bond cleavage (kcat/KM= 6.9 × 105M−1⋅s−1). The resulting C-terminally citrullinated peptides generated characteristic isotopic patterns in MALDI-TOF MS, and both a fragmentation producty1ion corresponding to citrulline (176.1030m/z) and diagnostic peak pairs in the extracted ion chromatograms of LC-MS/MS analysis. Using these signatures, we identified citrullination sites in protein arginine deiminase 4 (12 sites) and in fibrinogen (25 sites, two previously unknown). The unique mass spectral features of PTM-dependent proteolytic digest products promise a generalized PTM site-mapping strategy based on a toolbox of such mutant proteases, which are now accessible by laboratory evolution.

Towards the Standard-Module Approach to Disulfide-Linked Polypeptide Nanostructures. I. Methodological Prerequisites and Mass Spectrometric Characterization of the Test Two-Loop Structure

2003 ◽  
Vol 9 (2) ◽  
pp. 139-148 ◽  
Author(s):  
O.A. Mirgorodskaya ◽  
K.F. Haselmann ◽  
F. Kjeldsen ◽  
R.A. Zubarev ◽  
P. Roepstorff
Keyword(s):  
Mass Spectrometry ◽  
Disulfide Bonds ◽  
Bond Cleavage ◽  
Bond Formation ◽  
Peptide Bond ◽  
Biologically Active ◽  
Loop Structure ◽  
Partial Acid Hydrolysis ◽  
Peptide Bond Cleavage

Potentially biologically-active nanostructures can be created from single chains of unmodified peptides by cross-linking different regions of the chain by disulfide bonds and cleaving the chain at specified sites to obtain the final configuration. The availability of techniques for assembly and characterization of such structures was tested on a two-loop structure created from a 21-residue linear peptide. Directed intra-molecular disulfide bond formation was performed by inserting partial sequences favoring intra-molecular S–S bond formation (“loops”) separated by partial sequences disfavoring such a process (“spacers”) into the precursor sequence. Peptide bond cleavage by partial acid hydrolysis at specific sites (GG, NP/DP) inside the loops opened them; the same process in the spacer separated the loops. Synthesis, oxidation and bond cleavage were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF MS). The hydrolysis fragments of the produced nanostructures were characterized by tandem electrospray ionization Fourier transform mass spectrometry (ESI FT-MS) with collisional and electron capture dissociations. The latter technique was especially useful as it cleaves S–S bonds preferentially. The feasibility of the proposed synthesis approach and the adequacy of the analysis techniques for the test structure were demonstrated.

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