PMID- 29618278
OWN - NLM
STAT- MEDLINE
DCOM- 20190506
LR  - 20200306
IS  - 2472-6311 (Electronic)
IS  - 2472-6303 (Print)
IS  - 2472-6303 (Linking)
VI  - 23
IP  - 6
DP  - 2018 Dec
TI  - Automated Preparation of MS-Sensitive Fluorescently Labeled N-Glycans with a 
      Commercial Pipetting Robot.
PG  - 550-559
LID - 10.1177/2472630318762384 [doi]
AB  - N-Glycan analysis is routinely performed for biotherapeutic protein 
      characterization. A recently introduced N-glycan analysis kit using RapiFluor-MS 
      (RFMS) labeling provides time savings over reductive amination labeling methods 
      while also providing enhanced fluorescence (FLR) and mass spectrometry (MS) 
      responses. This article demonstrates the semiautomation of this kit using an 
      Andrew Alliance pipetting robot that promises further gains in productivity. This 
      robotic platform uses standard manual pipettors and an optically guided arm to 
      facilitate the automation of manual procedures. The manual RFMS protocol includes 
      two heating and cooling steps during protein denaturation and de-N-glycosylation. 
      However, the current Andrew Alliance automated platform cannot move reaction 
      tubes to and from different heating blocks. As a result, samples prepared using 
      the automated procedure remain in a computer-controlled Peltier effect heating 
      block, requiring reoptimization of denaturation and de-N-glycosylation 
      temperatures. Using hydrophilic interaction liquid chromatography to monitor the 
      RFMS-labeled glycan profiles, the authors demonstrated the reproducibility of the 
      automated protocol with percent relative standard deviations (RSDs) of 9%-19% for 
      the total area and 0.8%-20% for the relative areas of major and minor glycoforms. 
      Overall, the automated platform presented here proves to be a convenient and 
      reliable solution for N-glycan preparation and analysis.
FAU - Reed, Corey E
AU  - Reed CE
AD  - 1 Waters Corporation, Milford, MA, USA.
FAU - Fournier, Jennifer
AU  - Fournier J
AD  - 1 Waters Corporation, Milford, MA, USA.
FAU - Vamvoukas, Nikolaos
AU  - Vamvoukas N
AD  - 2 Andrew Alliance, Geneva, Switzerland.
FAU - Koza, Stephan M
AU  - Koza SM
AD  - 1 Waters Corporation, Milford, MA, USA.
LA  - eng
PT  - Journal Article
DEP - 20180404
PL  - United States
TA  - SLAS Technol
JT  - SLAS technology
JID - 101697564
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Polysaccharides)
RN  - 0 (Proteins)
SB  - IM
MH  - Automation, Laboratory/*methods
MH  - Fluorescent Dyes/metabolism
MH  - Mass Spectrometry/instrumentation/*methods
MH  - Polysaccharides/*analysis
MH  - Proteins/*chemistry
MH  - Reproducibility of Results
MH  - Robotics/*instrumentation
MH  - Staining and Labeling/instrumentation/*methods
PMC - PMC6249649
OTO - NOTNLM
OT  - N-glycan analysis
OT  - UPLC
OT  - automation
OT  - semiautomation
COIS- Declaration of Conflicting Interests: The authors disclosed the following 
      potential conflicts of interest with respect to the research, authorship, and/or 
      publication of this article: All authors are employees of Waters Corporation or 
      Andrew Alliance. Waters Corporation manufactures the GlycoWorks with RFMS kits 
      and liquid chromatography systems used in the research presented here. Andrew 
      Alliance manufactures the Andrew pipetting robot used in this research. The 
      authors declared no other potential conflicts of interest with respect to the 
      research, authorship, and/or publication of this article.
EDAT- 2018/04/06 06:00
MHDA- 2019/05/07 06:00
PMCR- 2018/11/22
CRDT- 2018/04/06 06:00
PHST- 2018/04/06 06:00 [pubmed]
PHST- 2019/05/07 06:00 [medline]
PHST- 2018/04/06 06:00 [entrez]
PHST- 2018/11/22 00:00 [pmc-release]
AID - 10.1177_2472630318762384 [pii]
AID - 10.1177/2472630318762384 [doi]
PST - ppublish
SO  - SLAS Technol. 2018 Dec;23(6):550-559. doi: 10.1177/2472630318762384. Epub 2018 
      Apr 4.