PMID- 33596068
OWN - NLM
STAT- MEDLINE
DCOM- 20211026
LR  - 20211026
IS  - 1879-1123 (Electronic)
IS  - 1044-0305 (Linking)
VI  - 32
IP  - 3
DP  - 2021 Mar 3
TI  - Electron Transfer Dissociation Parameter Optimization Using Design of Experiments 
      Increases Sequence Coverage of Monoclonal Antibodies.
PG  - 762-771
LID - 10.1021/jasms.0c00458 [doi]
AB  - Middle-down analysis of monoclonal antibodies (mAbs) by tandem mass spectrometry 
      (MS(2)) can provide detailed insight into their primary structure with minimal 
      sample preparation. The middle-down approach uses an enzyme to cleave mAbs into 
      Fc/2, LC, and Fd subunits that are then analyzed by reversed phase liquid 
      chromatography tandem mass spectrometry (RPLC-MS(2)). As maximum sequence 
      coverage is desired to obtain meaningful structural information at the subunit 
      level, a host of dissociation methods have been developed, and sometimes 
      combined, to bolster fragmentation and increase the number of identified 
      fragments. Here, we present a design of experiments (DOE) approach to optimize 
      MS(2) parameters, in particular those that may influence electron transfer 
      dissociation (ETD) efficiency to increase the sequence coverage of antibody 
      subunits. Applying this approach to the NIST monoclonal antibody standard 
      (NISTmAb) using three RPLC-MS(2) runs resulted in high sequence coverages of 67%, 
      67%, and 52% for Fc/2, LC, and Fd subunits, respectively. In addition, we apply 
      this DOE strategy to model the parameters required to maximize the number of 
      fragments produced in "low", "medium", and "high" mass ranges, which ultimately 
      resulted in even higher sequence coverages of NISTmAb subunits (75%, 78%, and 64% 
      for Fc/2, LC, and Fd subunits, respectively). The DOE approach provides high 
      sequence coverage percentages utilizing only one fragmentation method, ETD, and 
      could be extended to other state-of-the-art techniques that combine multiple 
      fragmentation mechanisms to increase coverage.
FAU - Cejkov, Milos
AU  - Cejkov M
AD  - Analytical Chemistry, Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River 
      Road, Tarrytown, New York 10591-6707, United States.
FAU - Greer, Tyler
AU  - Greer T
AD  - Analytical Chemistry, Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River 
      Road, Tarrytown, New York 10591-6707, United States.
FAU - Johnson, Reid O'Brien
AU  - Johnson RO
AD  - Analytical Chemistry, Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River 
      Road, Tarrytown, New York 10591-6707, United States.
FAU - Zheng, Xiaojing
AU  - Zheng X
AUID- ORCID: 0000-0002-7720-5149
AD  - Analytical Chemistry, Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River 
      Road, Tarrytown, New York 10591-6707, United States.
FAU - Li, Ning
AU  - Li N
AD  - Analytical Chemistry, Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River 
      Road, Tarrytown, New York 10591-6707, United States.
LA  - eng
PT  - Journal Article
DEP - 20210217
PL  - United States
TA  - J Am Soc Mass Spectrom
JT  - Journal of the American Society for Mass Spectrometry
JID - 9010412
RN  - 0 (Antibodies, Monoclonal)
RN  - 0 (Immunoglobulin Fab Fragments)
SB  - IM
MH  - Amino Acid Sequence
MH  - Antibodies, Monoclonal/*chemistry
MH  - Chromatography, Reverse-Phase/methods
MH  - Electrons
MH  - Immunoglobulin Fab Fragments/chemistry
MH  - Tandem Mass Spectrometry/methods
EDAT- 2021/02/18 06:00
MHDA- 2021/10/27 06:00
CRDT- 2021/02/17 17:08
PHST- 2021/02/18 06:00 [pubmed]
PHST- 2021/10/27 06:00 [medline]
PHST- 2021/02/17 17:08 [entrez]
AID - 10.1021/jasms.0c00458 [doi]
PST - ppublish
SO  - J Am Soc Mass Spectrom. 2021 Mar 3;32(3):762-771. doi: 10.1021/jasms.0c00458. 
      Epub 2021 Feb 17.