scholarly journals Mass spectrometry-based sequencing of the anti-FLAG-M2 antibody using multiple proteases and a dual fragmentation scheme

2021 ◽  
Author(s):  
Weiwei Peng ◽  
Matti F Pronker ◽  
Joost Snijder
Keyword(s):  
Monoclonal Antibody ◽  
De Novo ◽  
High Energy ◽  
De Novo Sequencing ◽  
Structural Basis ◽  
Sequence Information ◽  
Energy Collision ◽  
Sequence Coverage ◽  
High Energy Collision ◽  
Variable Regions

Antibody sequence information is crucial to understanding the structural basis for antigen binding and enables the use of antibodies as therapeutics and research tools. Here we demonstrate a method for direct de novo sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for de novo sequencing by LC-MS/MS in a bottom-up fashion. Furthermore, we apply a dual fragmentation scheme, using both stepped high-energy collision dissociation (stepped HCD) and electron transfer high-energy collision dissociation (EThcD) on all peptide precursors. The method achieves full sequence coverage of the monoclonal antibody Herceptin, with an accuracy of 98% in the variable regions. We applied the method to sequence the widely used anti-FLAG-M2 mouse monoclonal antibody, which we successfully validated by remodeling a high-resolution crystal structure of the Fab and demonstrating binding to a FLAG-tagged target protein in Western blot analysis. The method thus offers robust and reliable sequences of monoclonal antibodies.

Optimization of carbamylation conditions and study on the effects on the product ions of carbamylation and dual modification of the peptide by Q-TOF MS

2018 ◽  
Vol 24 (5) ◽  
pp. 384-396
Author(s):  
Cheng Guo ◽  
Xuefeng Guo ◽  
Lei Zhao ◽  
Dandan Chen ◽  
Jin Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
De Novo ◽  
De Novo Sequencing ◽  
Peptide Sequence ◽  
High Mass ◽  
Sequence Information ◽  
Amino Acid Residues ◽  
Sequence Coverage ◽  
Modification Method ◽  
Modified Peptides

Modified peptides fragmented by collision-induced dissociation can offer additional sequence information, which is beneficial for the de novo sequencing of peptides. Here, the model peptide VQGESNDLK was carbamylated. The optimal conditions were as follows: temperature of 90℃, pH of 7, and the time of 60 min. Then, we studied the b- and y-series ions of the native, carbamylated, and dual-modified peptides. The results were as follows. The short carbamylated peptides (≤10 amino acid residues) produced more b-series ions (including b1 ion). The long carbamylated peptides (>10 amino acid residues) produced additional b1 ion but fewer y-series ions (especially in the high-mass region). The short dual-modified peptides produced more b-series ions (including b1 ion) and more y-series ions, and their peptide sequence coverage was almost 100%. The long dual-modified peptides produce b1 ion and more y-series ions, and their peptide sequence coverage was nearly above 90%. Therefore, both carbamylation and the dual modification method could be used to identify the N-terminal amino acid, and the dual modification method was also excellent for the de novo sequencing of the tryptic peptides.

Hb Neuilly-Sur-Marne, a New Human Hemoglobin Variant with Ser-Asp-Leu Inserted between α86(F7) Leu and α87(F8) His: Characterization by High-Energy Collision-Induced Dissociation Liquid Secondary Ion Mass Spectrometry and Low Energy Collision-Induced Dissociation Tandem Mass Spectrometry in An Ion Trap Fitted with a Nanospray Ionization Source

2000 ◽  
Vol 6 (2) ◽  
pp. 205-211 ◽  
Author(s):  
Danielle Promé ◽  
Jean-Claude Promé ◽  
Henri Wajcman ◽  
Jean Riou ◽  
Frédéric Galactéros ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Secondary Ion Mass Spectrometry ◽  
Ion Trap ◽  
High Energy ◽  
Sequence Information ◽  
Energy Collision ◽  
Ionization Source ◽  
Nanospray Ionization ◽  
Singly Charged ◽  
Secondary Ion

Hemoglobin (Hb) Neuilly-sur-Marne is a new α-chain variant found during a systematic screening. Electrospray mass measurements showed the presence of an abnormal α-chain displaying a shift of +315 u relative to the normal value. Tryptic cleavage of this chain and molecular weight determination of the peptides indicated that the 315 u shift was located into the αT-9 peptide, the molecular weight of which is higher than 3000 Da. High-energy collision spectra of MH+ ions generated by liquid secondary ion mass spectrometry from the normal and abnormal αT-9 afforded mainly amino-terminal containing ions. They indicated that these two peptides have an identical amino acid sequence from their 1st to 25th residues, the mass increase being thus located beyond this point. Too few ions were formed to establish reliably the sequence forward. It was hypothesized that this mass shift could result from a repeated sequence since the sum of the mass of the three residues—leucine, serine and aspartic acid—preceding position 25 is exactly 315 u. To get sequence information above position 25, decomposition of multicharged species was attempted. An ion trap fitted with a nanospray ionization source was used. It produced mainly triply- and quadruply-charged ions. Decomposition of the triply-charged ion afforded a series of singly-charged Y-ions in the expected region, giving a readily interpretable sequence. It confirmed the insertion of a Ser-Asp-Leu sequence above position 25. Surprisingly, decomposition of the quadruply-charged molecular ion gave too few ions to provide sequence information in the expected region. Spectra were dominated by some multicharged Y ions arising from cleavages close to the amino end. Tandem mass spectrometry experiments were performed on the abundant Y303+ ion and produced again a singly-charged Y ion series in the suitable domain which confirmed the above result. In Hb Neuilly-sur Marne this insertion of the Ser-Asp-Leu residues. between positions α-86 and α-87 is very likely due to a slipped strand mispairing mechanism.

scholarly journals FRACTAL GEOMETRY AND QUARK-GLUON PLASMA PHYSICS

2020 ◽  
Vol 2 ◽  
pp. 1
Author(s):  
N. G. Antoniou
Keyword(s):  
Plasma Physics ◽  
Quark Gluon Plasma ◽  
Fractal Geometry ◽  
Gluon Plasma ◽  
High Energy ◽  
Energy Collision ◽  
High Energy Collision ◽  
One Dimensional ◽  
Interaction Dynamics ◽  
The One

Incorporating fractal geometry in the Regge-Mueller approach to strong interaction dynamics one may formulate a model for the one-dimensional critical sector of the hadronic 5-matrix in a high energy collision. A non conventional component of the correlation functions in rapidity space is obtained, the phenomenological implications of which are related with the intermittency effects in quark-gluon plasma physics.

High-energy collision-induced dissociation of phosphopeptides using a multi-turn tandem time-of-flight mass spectrometer ‘MULTUM-TOF/TOF’

2008 ◽  
Vol 43 (4) ◽  
pp. 535-537 ◽  
Author(s):  
Shuichi Shimma ◽  
Hirofumi Nagao ◽  
Anastassios E. Giannakopulos ◽  
Shigeo Hayakawa ◽  
Kunio Awazu ◽  
...  
Keyword(s):  
Mass Spectrometer ◽  
Time Of Flight ◽  
High Energy ◽  
Collision Induced Dissociation ◽  
Energy Collision ◽  
High Energy Collision ◽  
Flight Mass Spectrometer

scholarly journals Structural elucidation of O-linked glycopeptides by high energy collision-induced dissociation

1996 ◽  
Vol 7 (4) ◽  
pp. 319-328 ◽  
Author(s):  
K. F. Medzihradszkyaff ◽  
B. L. Gillece-Castroaff ◽  
R. R. Townsendaff ◽  
A. L. Burlingameaff ◽  
M. R. Hardyaff
Keyword(s):  
High Energy ◽  
Structural Elucidation ◽  
Collision Induced Dissociation ◽  
Energy Collision ◽  
High Energy Collision

Subcascade formation and defect cluster size scaling in high-energy collision events in metals

2016 ◽  
Vol 115 (2) ◽  
pp. 26001 ◽  
Author(s):  
A. De Backer ◽  
A. E. Sand ◽  
K. Nordlund ◽  
L. Luneville ◽  
D. Simeone ◽  
...  
Keyword(s):  
Cluster Size ◽  
High Energy ◽  
Defect Cluster ◽  
Energy Collision ◽  
High Energy Collision ◽  
Size Scaling
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