42 Buyers’ Guide 2021
1000 HeLa digests, each followed by a blank and a cytochrome c digest injection, which results in a total number of 3526 injections on a single µ-pillar array column. The HeLa digest separation was performed in a 30 minute gradient, 60 minute cycle time, with a flow rate of 1 µL/min. The mobile phases A and B were prepared at the start of the experiment, and not refreshed for the duration of the experiment. Despite the degradation of the mobile phases, the repeatability of the HeLa digest separation is outstanding [17].
Figure 2. Column to column reproducibility over seven different µPAC™ columns, 500 fmol/µL of digested cytochrome c injected on each column.
Moreover, with the increasing importance of protein-based drugs, proteomics workflows are finding their way into pharmaceutical and biotech laboratories, with peptide mapping becoming an essential part in the discovery and development of therapeutic monoclonal antibody (mAb) or antibody-drug conjugate (ADC) targets. These molecules are large (approximately 150 kDa) and heterogeneous, differing in post-translational modifications, amino acid structures and higher order structures [18]. With larger numbers of original mAb drugs running out of patent, biosimilar products are expected to become available. As the name implies, they will have to be highly similar but not identical. To characterise and monitor this similarity, highly sensitive and high resolution LC/MS are required. Figure 4 shows an example of the comparison of the original Remicade drug versus a candidate biosimilar, using a tryptic digest peptide map and clearly showing five distinct differences in the total compound chromatograms.
Figure 3. µ-pillar array column robustness. UV chromatograms obtained for the separation of 100 ng tryptic digested HeLa cells. Injections 1 to 1000 are displayed at a 100 injections interval. Injection volume 1 µL; flow rate 1 µL/min; gradient conditions 1-50% B in 30 min; mobile phase A H2 H2O+80% ACN+0.1% TFA; column temperature 35°C; UV detection 214 nm.
O+0.1% TFA / B 20%
But µ-pillar array columns are not only restricted to peptide mapping applications. Metabolomics and lipidomics researchers are also performing more sample restricted experiments. Although nanoLC/MS might not always be their first choice, despite the sensitivity that can be achieved, reduced column IDs are being investigated. Promising results have been achieved at low microliter per minute flow rates, typically performed using 300 µm ID columns [19]. However, these columns would be packed with the same stationary phase as their nanoLC counterparts, with the same challenges as described above.
Figure 4. LC-MS total compound chromatogram of the tryptic digest of a Remicade original drug (bottom trace) and a candidate biosimilar (upper trace). Distinct differences in the chromatograms are labelled 1-5.
Again, µ-pillar array columns can be used here as well. For instance, sample complexity in lipidomics is quite considerable, with the LIPID MAPS Structure Database consisting of just under 45000 unique lipid structures. With µ-pillar array column lengths of 200 cm, these columns are ideal to take on this complexity, as is demonstrated in Figure 5 where a human blood plasma lipid extract was analysed in a 60 minute gradient [20]. The upper trace
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