Automated mass correction and data interpretation for protein open-access liquid chromatography-mass spectrometry.
Characterization of recombinant protein purification fractions and final products by liquid chromatography-mass spectrometry (LC/MS) are requested more frequently each year. A protein open-access (OA) LC/MS system was developed in our laboratory to meet this demand. This paper compares the system that we originally implemented in our facilities in 2003 to the one now in use, and discusses, in more detail, recent enhancements that have improved its robustness, reliability, and data reporting capabilities. The system utilizes instruments equipped with reversed-phase chromatography and an orthogonal accelerated time-of-flight mass spectrometer fitted with an electrospray source. Sample analysis requests are accomplished using a simple form on a web-enabled laboratory information management system (LIMS). This distributed form is accessible from any intranet-connected company desktop computer. Automated data acquisition and processing are performed using a combination of in-house (OA-Self Service, OA-Monitor, and OA-Analysis Engine) and vendor-supplied programs (AutoLynx, and OpenLynx) located on acquisition computers and off-line processing workstations. Analysis results are then reported via the same web-based LIMS. Also presented are solutions to problems not addressed on commercially available, small-molecule OA-LC/MS systems. These include automated transforming of mass-to-charge (m/z) spectra to mass spectra and automated data interpretation that considers minor variants to the protein sequence-such as common post-translational modifications (PTMs). Currently, our protein OA-LC/MS platform runs on five LC/MS instruments located in three separate GlaxoSmithKline R&D sites in the US and UK. To date, more than 8000 protein OA-LC/MS samples have been analyzed. With these user friendly and highly automated OA systems in place, mass spectrometry plays a key role in assessing the quality of recombinant proteins, either produced at our facilities or bought from external sources, without dedicating extensive amounts of analyst resource.
Wagner
Craig D
CD
Molecular Discovery Research, GlaxoSmithKline, Research Triangle Park, NC 27709, USA.
Hall
John T
JT
White
Wendy L
WL
Miller
Luke A D
LA
Williams
Jon D
JD
D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry*; D002364:Caseins / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D001330:Electronic Data Processing; D019451:Information Management; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
D002364:Caseins; D011994:Recombinant Proteins; D002256:Carbonic Anhydrases