GEN-MKT-18-7897-A
Mar 18, 2016 | Blogs, Life Science Research | 0 comments
If you have a TripleTOF® System and you’re using SWATH Acquisition for your quantitative proteomics experiments, you’ve made the right choice. SWATH is a data independent workflow that enables data to be acquired for every detectable analyte in a complex sample. You create a digital map of the sample that can be mined for new information any time new biological insights are hypothesized. It’s all there!
Since the introduction of the workflow at HUPO 2010 in Sydney, Australia, we have been working to further improve the workflow. Optimizations in data acquisition such as smaller and variable Q1 window widths and increased instrument dynamic range have provided increased depth of coverage for your proteomics sample, without compromising reproducibility or data quality.
But what about data processing? Can we improve even further?
The answer is yes! Typically an ion library is used for targeted processing of SWATH data for peptide and protein identification and quantitation. The ion library contains the masses of the peptide ions, sequence-specific fragment ions, relative fragment intensities, and relative retention times, and is easily generated by performing a simple data-dependent acquisition and database search.
In this study, scientists from SCIEX demonstrate that for biological systems that will be studied repeatedly, it is worth taking the extra time to create a deeper ion library as much more information can be extracted from the SWATH acquisition data.
For example, SWATH replicates of a HEK human cell lysate were acquired and processed using three different libraries:
Figure 1. The Impact of Deeper Ion Libraries on Extraction of Quantitative Data from Human Cell Lysate SWATH® Acquisition Data.
As shown in Figure 1, a 118% gain in quantified proteins was observed using a simple ion library (1D HEK) and an extensive ion library (PHL). And the quality and reproducibility of the quantitation are maintained even into the low abundant protein/peptide regime.
A researcher can balance the library generation time with the depth of coverage needed. The three libraries outlined here took increasingly more time to generate. A simple IDA experiment can take only a matter of hours. A 2D fractionation followed by LC-MS/MS on each fraction can be performed in a matter of days. And some groups have invested significantly more time in library generation using multiple cell types and a large degree of fractionation to cover as much of the proteome as possible.
This work also highlights the superior dynamic range of quantitative information that is present in a SWATH Acquisition data file over the traditional data dependent approaches for quantitative proteomics. To read more details of this work, and to see what size
To read more details of this work, and to see what size ion library would make the most sense for your biological system of study, download the full technical note.
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.
Posted by
You must be logged in to post a comment.
Share this post with your network