GEN-MKT-18-7897-A
Sep 15, 2015 | Blogs, Life Science Research, Proteomics | 0 comments
Frankly, who has the time? With so many samples to prepare, day after day, week after week, month after month, there’s time for little else. And those repetitive tasks of pipetting over and over again, just keep, well, repeating.
It’s time to automate your sample prep! In this technical note, see how researchers reduce day-to-day variability resulting from manual, time-consuming, and multi-step protocols such as the protein denaturation, reduction, alkylation and digestion steps which precede LC/MS analysis. Here, the researchers use a Biomek NXP Span-8 Workstation and protein preparation kits with ready-to-use reagents to automate the entire workflow. Then, they tested the automated workflow for their quantitative proteomics studies.
First, the reproducibility of the digestion protocol alone was tested. As shown in the technical note, very high reproducibility was obtained, with about 90% of peptides monitored by LC/MS having digestion reproducibility below 10% CVs. Next, the digestion protocol was transferred to the Biomek NXP Span-8 Workstation. Many of the proteotypic peptides analyzed showed very high reproducibility with %CVs ~ 2-3%. As expected, for both the manual and automated protocol some peptides showed higher variability than others from the same protein, reflecting inherent digestion heterogeneity. When developing assays for peptide/protein quantitation, it’s important to assess the variability in digestion reproducibility in order to choose the highest performing peptides. Automation of this step can greatly simplify the assessment.
Finally, the researchers tested the reproducibility across different labs and workstations, and across multiple days. The results show very similar cumulative reproducibility curves with more than 80% of peptides monitored having CVs below 10%, demonstrating that the automation method and protocol were very robust and transferable.
To learn more, download the full technical note and see how the Biomek Workstation with optimized protein preparation kits can help you save time while providing reproducible sample preparation today.
Now, what will you do with all that free time?
Finding the right information shouldn’t slow you down. Whether you’re troubleshooting your mass spec, learning something new, or optimizing performance, access to the right resources at the right moment makes all the difference.
As an analytical strategy, middle-down mass spectrometry (MS) workflows characterize biotherapeutic proteins by analyzing large, digested protein fragments or defined subunits, rather than fully intact proteins (top-down) or digested peptides (bottom-up). A middle-down strategy combines the strengths of top-down and bottom-up approaches by delivering high sequence coverage and structural specificity while maintaining relatively simple sample preparation. In practice, middle-down analysis enables accurate mass measurement, rapid sequence confirmation, and localization of key post-translational modifications (PTMs) on protein subunits that are directly relevant to product quality.
In biopharmaceutical development, sequence variants (SV) are considered an inherent risk of producing complex proteins in living systems. Sequence variants are unintended changes to the amino acid sequence of a biotherapeutic and can be caused by errors in transcription or translation in the host cell, or cell culture and process conditions. Detailed analysis of SVs is important in process and product development to ensure the drug’s safety and efficacy. Even low‑level sequence variants can have significant implications for product quality, safety, and efficacy, making their accurate detection and characterization a critical requirement across development, process optimization, and regulatory submission.
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