Proteomics & Biopharma – Different Foci in Protein Analysis Need Specific Demands on Instrumentation

M. Macht, Bruker Daltonik GmbH, Bremen, Germany
W. Jabs, Bruker Daltonik Gmbh, Bremen, Germany
A. Resemann, Bruker Daltonik Gmbh, Bremen, Germany
A. Wiechmann, Bruker Daltonik Gmbh, Bremen, Germany
W. Imhoff, Bruker Daltonik Gmbh, Bremen, Germany
W. Evers, Bruker Daltonik Gmbh, Bremen, Germany
C. Evans, Bruker Daltonik Gmbh, Bremen, Germany
R. Hartmer, Bruker Daltonik Gmbh, Bremen, Germany
D. Suckau, Bruker Daltonik Gmbh, Bremen, Germany
S. Kaspar, Bruker Daltonik Gmbh, Bremen, Germany
M. Lubeck, Bruker Daltonik Gmbh, Bremen, Germany
A. Michalski, Bruker Daltonik Gmbh, Bremen, Germany
M. Andersen, Bruker Daltonik Gmbh, Bremen, Germany
P.-O. Schmit, Bruker Daltonique, Paris, France
E. Wagner-Rousset, Centre D’immunologie Pierre Fabre, Saint Julien En Genevois, France
A. Beck, Centre D’immunologie Pierre Fabre, Saint Julien En Genevois, France

Although shotgun proteomics and analysis of biopharmaceuticals, typically monoclonal antibodies (mAbs), deal with proteins, the analytical questioning is significantly different. One of the most important workflows for mAbs characterization is reduction of the antibody to give free light (LC) and heavy chains (HC) which are then detected by LC-MS. The workflow is commonly applied to verify the amino-acid sequence and to determine post-translational modifications. The purpose of Shotgun proteomics is to provide quantitative information on the protein content of a biological sample, e.g. tryptic digests of whole cell lysates. The very high dynamic range of these samples remains the major challenge.

Deploying a significantly improved maXisII ultrahigh resolution ESI QTOF extends the specifity of workflow to characterize mABs, providing improved resolving capability to isotopically resolve antibody HCs on a liquid chromatographic timescale. After reduction, LC-MS runs were recorded for natalizumab, a therapeutic antibodies (IgG4). Monoisotopic masses were determined after Maximum Entropy deconvolution and peak picking (SNAP algorithm) and applied to the verification of aminoacid sequences. Two different sequences were evaluated, differing by three amino acids resulting in a 2Da difference for the HC. The application of this workflow to distinguish, e.g., asparagine deamidation from aspartate isomerization on the HC level is discussed.

In a shotgun proteomics experiment, the quantification strategy and the instrumentation used is key for successfully revealing biological information. High-performance QTOFs, such as the impactII with 50,000 resolution over entire relevant mass range and low ppm mass accuracy provide excellent results. Method used automatically adapts the MS/MS acquisition speed to precursor intensity. System global performances, quant accuracy and dynamic range, was assayed using yeast as background, spiked with UPS2 in a ratio 1:2. LC-MS/MS runs were analyzed with MaxQuant software. Results show excellent quantitation of the UPS2 proteins over 4orders of magnitude and with CV values below 10%.