ProMass 6 for Xcalibur and MassLynx Release! What is new?

Mass spectrometry is a highly useful tool for the characterization of messenger RNA (mRNA). PolyA tail length is an important characteristic that affects both the stability and translation efficiency of the mRNA. A unique feature of polyA tails is the heterogeneity in their length. Accurately determining a polyA tail average length and distribution in a sample is critical for understanding the functionality and/or quality control in mRNA production. PolyA tails are typically heterogeneous, which results in complex looking spectra, as shown below.

With ProMass processing this complex spectrum is transformed into the easy-to-interperet deconvoluted spectrum shown below.

ProMass 6 now has a data processing workflow specifically for polyA tails that will provide the user with information such as PolyA tail distribution and the average PolyA tail length. When the PolyA workflow is utilized in ProMass processing, ProMass will automatically generate a PolyA tail report as shown below.

Bottom-up mass spectrometry is a tried-and-true strategy employed for sequence confirmation of large proteins and/or oligonucleotides. This methodology involves protein or oligonucleotide digestion with an enzyme that results in shorter sequences which are more readily analyzed by LCMS. The analyzed digestion products are matched against the theoretical masses for expected digestion fragments and are mapped to confirm the identity of full-length target sequence. This type of data processing can be labor-intensive if done manually. ProMass 6’s new enzyme digestion feature streamlines and automates bottom-up mass spectrometry data processing.

The example below illustrates data processing of a tryptic digest of Waters intact mAb standard using ProMass 6’s Enzyme Digestion and Mapping feature. ProMass processing generates an interactive sequence coverage map in a single report and a summary table to indicate the coverage percentage.

ProMass 6’s new Enzyme Digestion and Mapping feature includes several enzymes pre-defined and ready to use. This includes trypsin, chymotrypsin, GluC, V8, and AspN for proteins as well as both RNase T1 and RNase 4 for oligonucleotide samples. Users can also define their own enzymes if they would like.

Check out our white paper that highlights the functionality of the new Oligo Sequence Confirmation features in ProMass 6 HR!

There are several new features added to the OligoSeq sequence confirmation tool.

The ability to customize which oligonucleotide MS/MS fragments to match as well as match internal MS/MS fragments that may be present in the data.

Added an Extended Report option that performs a base swap analysis on the data and provides a score for the candidate sequence(s).

Adding multiple target sequences with an Extended Report will perform a comparative analysis of the provided sequences. This is great for differentiating MS/MS data of ambiguous sequences produced from bottom-up mRNA sequencing approaches.

ProMass 6 HR OligoSeq tool now supports the ability for users to define unique species that undergo similar MS/MS fragmentation to oligonucleotides, such as phosphorodiamidate morpholino oligomers (PMOs), for sequence confirmation.

Positive Probability Limited (PPL) methods enable users to accurately de-isotope high resolution mass spectra. Previous versions of ProMass included standardized PPL methods for generic oligonucleotide or protein de-isotoping. However, users would still need to manually generate PPL methods when processing samples with unique chemical compositions (such as phosphorothioated nucleic acids) to minimize de-isotoping artifacts. ProMass HR 6 introduces a new feature to simplify data processing in these situations, PPL Auto Mode. If selected, PPL Processing Parameters will automatically be adjusted based on user input/output mass ranges, input sequence(s), or molecular formula.

With PPL Auto Mode processing high resolution mass spectra has never been easier.

ProMass 6 now has a pre-filter feature that can be used to reject baseline noise in larger datasets which can dramatically improve processing speeds (2x or more).

When processing samples with ProMass to calculate Drug-Antibody Ratios (DAR), reports generated by ProMass 6 include a graphical representation of DAR distribution. This graphics are interactive and provide direct links to the spectra and data that support the relevant portion of the graphic.

Automatically processed LC-MS data now produces a composite spectrum view providing users with a unique global view of the entire data set.

This is particularly useful for DAR and polyA data sets.

ProMass 6 introduces a new interactive viewer that illustrates the portions of a sequence identified in a sample analysis. In addition to enzyme digest analysis, this feature is particularly useful when processing impurity profiling data where there are truncated sequences. The example shown below illustrates the summary viewer from data processed with ProMass’ ladder summary illustrating the analysis of oligonucleotide impurities with truncated sequences from the 5′ end.

This viewer is produced whenever a sequence ladder is calculated. In addition to impurity profiling applications, the viewer makes it even easier to readily identify regions of a sequence that are susceptible to degradation during metabolite identification studies.

One of the great features of ProMass is the ability to share the interactive reports with colleagues or collaborators without the requirement of additional software other than a web browser to view the data. ProMass 6 reports now include an interactive chromatogram viewer which enables any user to view the peak integrations and the hyperlinked spectra for peaks of interest directly within the web browser-based ProMass reports.