Protein mass measurement combined with mass spectrometric sequencing of protein digests for detection and characterization of protein modifications1

2006 ◽  
Vol 84 (7) ◽  
pp. 986-997 ◽  
Author(s):  
Chengjie Ji ◽  
Zhengping Wang ◽  
Liang Li
Keyword(s):  
Mass Spectrometry ◽  
Gel Electrophoresis ◽  
Protein Modification ◽  
Mass Spectrometric ◽  
Maldi Ms ◽  
Intact Protein ◽  
Protein Mass ◽  
Cell Extracts ◽  
Peptide Mass

A method for the characterization of modifications of low molecular weight proteins (<20 kDa) extracted from a microorganism based on the use of multiple separation tools and mass spectrometric techniques is described. In this method, intact proteins from cell extracts are first separated and fractionated by liquid chromatography (LC). Individual fractions are then analyzed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) to provide intact protein mass information. The fractions are further characterized by using trypsin digestion and LC electrospray ionization (ESI) MS/MS analysis of the resultant peptides to identify the proteins. Gel electrophoresis of a fraction is also carried out to estimate the molecular masses of the proteins. The gel bands are identified by in-gel digestion and peptide mass mapping and sequencing using MALDI-MS and MALDI-MS/MS. The combined information generated from these experiments is interpreted for detecting and characterizing modified proteins. This method has been developed and applied to the analysis of posttranslational modifications (PTMs) of low-mass proteins (5–20 kDa) extracted from a relatively well-characterized microorganism, Escherichia coli. Using this method, not only previously reported PTMs involving acetylation, methylation, oxidation, and the removal of signal peptides, but also two novel PTMs, namely loss of N-terminal Met-Thr-Met (MTM) and hydroxylation of arginine, were identified. It is envisaged that this method should be applicable to other relatively simple microorganisms for the discovery of new PTMs.Key words: top-down proteomics, protein modification, HPLC, gel electrophoresis, tandem mass spectrometry.

scholarly journals Characterization of [3H]palmitate- and [3H]ethanolamine-labelled proteins in the multicellular parasitic trematode Schistosoma mansoni

1988 ◽  
Vol 254 (2) ◽  
pp. 419-426 ◽  
Author(s):  
P M Wiest ◽  
E J Tisdale ◽  
W L Roberts ◽  
T L Rosenberry ◽  
A A F Mahmoud ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Free Amino ◽  
Protein Modification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Reductive Methylation ◽  
Amide Linkage

Biosynthetic labelling experiments with cercariae and schistosomula of the multicellular parasitic trematode Schistosoma mansoni were performed to determine whether [3H]palmitate or [3H]ethanolamine was incorporated into proteins. Parasites incorporated [3H]palmitate into numerous proteins, as judged by SDS/polyacrylamide-gel electrophoresis and fluorography. The radiolabel was resistant to extraction with chloroform, but sensitive to alkaline hydrolysis, indicating the presence of an ester bond. Further investigation of the major 22 kDa [3H]palmitate-labelled species showed that the label could be recovered in a Pronase fragment which bound detergent and had an apparent molecular mass of 1200 Da as determined by gel filtration on Sephadex LH-20. Schistosomula incubated with [3H]ethanolamine for up to 24 h incorporated this precursor into several proteins; labelled Pronase fragments recovered from the three most intensely labelled proteins were hydrophilic and had a molecular mass of approx. 200 Da. Furthermore, reductive methylation of such fragments showed that the [3H]ethanolamine bears a free amino group, indicating the lack of an amide linkage. We also evaluated the effect of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: [3H]palmitate-labelled proteins of schistosomula and surface-iodinated proteins were resistant to hydrolysis with this enzyme. In conclusion, [3H]palmitate and [3H]ethanolamine are incorporated into distinct proteins of cercariae and schistosomula which do not bear glycophospholipid anchors. The [3H]ethanolamine-labelled proteins represent a novel variety of protein modification.

scholarly journals Multilevel characterization of antibody-ligand conjugates by CESI-MS

2020 ◽  
Vol 20 ◽  
Author(s):  
Bryan Fonslow ◽  
Gabor Jarvas ◽  
Marton Szigeti ◽  
Andras Guttman
Keyword(s):  
Mass Spectrometry ◽  
Capillary Electrophoresis ◽  
Peptide Mapping ◽  
Site Occupancy ◽  
Intact Protein ◽  
Ionization Mass ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug

Aims: Demonstrating the capabilities of our new capillary electrophoresis – mass spectrometry method, which facilitates highly accurate relative quantitation of modification site occupancy of antibody-ligand (e.g., antibody-drug) conjugates. Background: Antibody-drug conjugates play important roles in medical discovery for imaging and therapeutic intervention. The localization and stoichiometry of the conjugation can affect the orientation, selectivity, specificity, and strength of molecular interactions, influencing biochemical function. Objective: To demonstrate the option to analyze the localization and stoichiometry of antibody-ligand conjugates by using essentially the same method at all levels including ligand infusion, peptide mapping, as well as reduced and intact protein analysis. Materials and Methods: Capillary electrophoresis coupled to electrospray ionization mass spectrometry was used to analyze the antibodyligand conjugates. Results: We identified three prevalent ligand conjugation sites with estimated stoichiometries of 73, 14, and 6% and an average ligand-antibody ratio of 1.37, illustrating the capabilities of CE-ESI-MS for rapid and efficient characterization of antibody-drug conjugates. Conclusion: The developed multilevel analytical method offers a comprehensive way to determine the localization and stoichiometry of antibody-drug conjugates for molecular medicinal applications. In addition, a significant advantage of the reported approach is that small, hydrophilic, unmodified peptides well separated from the neutrals, which is not common with other liquid phase separation methods such as LC.

Approach to the profiling and characterization of influenza vaccine constituents by the combined use of size-exclusion chromatography, gel electrophoresis and mass spectrometry

Biologicals ◽  
2010 ◽  
Vol 38 (2) ◽  
pp. 294-302 ◽  
Author(s):  
Virginia Garcia-Cañas ◽  
Barry Lorbetskie ◽  
Terry D. Cyr ◽  
Mary A. Hefford ◽  
Sophie Smith ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Influenza Vaccine ◽  
Size Exclusion Chromatography ◽  
Gel Electrophoresis ◽  
Size Exclusion ◽  
Combined Use ◽  
Exclusion Chromatography
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