- This application note describes a vendor neutral workflow for the analysis of intact therapeutic proteins using top-down fragmentation.
- The Intact Mass™ workflow in Byos® now provides automatic MS/MS spectral annotation, in addition to MS level deconvolution and identification. This offers significant advantages in decreasing the time burden required for data analysis over traditional bottom-up approaches.
- We demonstrate the benefit of the Intact Mass workflow for rapid and reliable intact mass deconvolution and MS/MS fragment annotation using high resolution mass spectrometry.
Monoclonal antibodies (mAbs) are important therapeutic glycoproteins, but their large size and structural complexity make them difficult to rapidly characterize. Mass spectrometry (MS) provides a powerful tool for the characterization of mAbs as well as other therapeutic proteins. In conventional "bottom-up" approaches, proteins are enzymatically digested, resulting in smaller peptides that can be easily analyzed with mass spectrometers. However, this technique encounters various challenges, such as development of the digestion protocol, separation of digest products, interpretation of MS/MS spectra, and the need for efficient data analysis and reporting.
Advancements in instrumentation and protein fragmentation techniques have enabled the development of "top-down" MS analysis. This methodology involves the fragmentation of intact protein ions, allowing for a more detailed structural characterization of the protein while maintaining complete sequence coverage.
Deconvolution of electrospray ionization mass spectra is typically required for intact / sub-unit protein-level MS experiments to obtain neutral masses.
Recent enhancements to the intact workflows (Intact, Reduced and ADC) now enable the reading and annotation of MS/MS spectra, providing sequence confirmation at the intact level.
Simplification of intact profiles can be achieved by creating smaller mAb fragments by digestion with the immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS), followed by reduction with dithiothreitol (DTT). IdeS digestion produces three subunits, each approximately 25 kDa (LC, Fd' and Fc/2). This middle-down approach is advantageous as it is quick, informative, and cost-effective in terms of materials.
This application note provides an overview to the updated intact workflows within Byos. A subunit analysis of NIST mAb was used to illustrate the software’s ability to simultaneously identify subunits (including glycoforms) at the intact level and and verify the primary sequence through the annotation of MS/MS spectra.
NIST mAb was digested with IdeS endopeptidase that cleaves the heavy chains below the hinge region, generating F(ab')2 and Fc fragments. After reduction of disulfide bonds, the three antibody domains (LC, Fd, and Fc/2) can be released for ESI-LCMS characterization (Figure 1).
Figure 1. Schematic of a mAb enzymatically and chemically cleaved into LC, Fd' and Fc/2 sub-units.
Analysis was performed using a high-resolution analytical platform consisting of a Thermo Scientific™ Dionex™ UHPLC and Thermo Scientific™ Q Exactive™ Elite Hybrid Quadrupole-Orbitrap™ mass spectrometer.
Data were analyzed using the Intact Mass workflow in Byos.
The top-down part of the intact workflow is straightforward to set-up and involves setting MS and MS/MS match tolerances (Figure 2b).
Figure 2a. Intact Workflow - Sequences and Masses tab.
When processing data, the user determines both MS and MS/MS Match tolerance for deconvoluted mass assignment and fragment annotation respectively.
Figure 2b. Match tolerance and MS2 tolerance settings (Zoomed-in view under Delta masses table).
IdeS Digestion Sequences
NIST protein sequences were added by drag & dropping the FASTA files into the workflow. Users can either define individual sequences for the reduced IdeS digest sub-units (Fc/2, Fd and LC), or enter full HC and LC sequences and define a specific clipping site as show in Figure 3.
Figure 3. Sequence combination and clipping option - Complete HC and LC sequences were selected in the sequence combination box. The clipping function was enabled and directed to clip between GG.
TIP: The workflow can be saved and renamed as IdeS with embedded sequences, clipping, delta masses and deconvolution settings ready to use with new raw data files.
Generating both MS and MS/MS level results from an intact analysis is challenging because of the differences in data output. The intact workflow in Byos now processes both intact MS and top-down MS/MS data. The annotated MS/MS plot annotation is presented alongside the deconvoluted intact spectrum and provides direct sequence annotation of the data set. Figure 4 shows the user interface which contains many tools and visualizations for data analysis, including annotated MS/MS spectra.
Figure 4. Intact Mass top-down user interface
Another view is the trace plot (Figure 5), which illustrates the total ion chromatogram (TIC) featuring three peaks representing the Fc/2, LC, and Fd NIST mAb subunits.
Figure 5. TIC Chromatogram of IdeS digested NIST mAb
The MS1 view (Figure 6) displays the summed m/z plot for the first peak in the TIC. This data remains unprocessed, aside from the summation of individual scans. Throughout data acquisition, five precursor charge states were selected for each subunit within their corresponding charge state distribution. These were isolated by the quadrupole within the 960-1180 m/z window and subjected to ETD fragmentation. Ionization of the reduced LC and HC from NIST mAb in denaturing conditions yields two charge state envelopes from 22+ to 26+ for the LC, from 22+ to 26+ for the Reduced Fc/2, from 30+ to 65+ for the Fc
MS/MS scans were acquired at 120,000 resolving power (at m/z 200) with an isolation width of 5 m/z.
Figure 6. MS1 TIC Plot of the 5 most abundant ions from the quadrupole isolation window
Figure 7. Deconvoluted mass spectrum of IdeS reduced NIST mAb Fc/2 glycoforms.
Figure 8. MS/MS Plot with top-down NIST mAb sub-unit ETD fragments annotation.
MS/MS spectra are not deconvoluted
The standard approach to top-down proteomics includes preprocessing the MS/MS spectra through de-charging and de-isotoping of peaks before comparing the spectrum to theoretical product ions derived from potential protein sequences. This approach has a number of advantages: it simplifies complex spectra, eases the task of matching peaks to theoretical ions, and facilitates dynamic programming (for example, for gapped alignment or sequence tagging). This approach also suffers from a number of disadvantages: de-charging and de-isotoping make frequent errors, remove potentially valuable information (namely product ion charge states), and preclude the use of low-resolution mass analyzers for measuring product ions. For example, Figure 9 shows two overlapping high-resolution fragments spanning around 1020m/z. The MS/MS annotation unambiguously assigns the z28 (+3) and c29 (+3).
NOTE: A new feature has been added for MS/MS annotation. The heights of the bars for the annotated peaks reflects the integral intensity of the isotope envelope of the putative ion.
*The annotation of integrated intensity can also be disabled through the annotations option menu if needed*
Figure 9. MS/MS annotation feature for overlapping fragments resulting in unambiguous annotation of experimental z28 and c29 (+3) charge states fragments spectrum.
Byos contains a variety of preloaded report templates, which enable users to generate multi-tab reports containing pivot tables, charts and plots.
The default intact report contains a summary tab with sample and processing data information (alongside meta LC-MS/MS acquisition data) and pivot tables with intensity and relative intensity options (Figure 10).
Figure 10a. Default Intact Mass Report - Pivot table showing the relative intensity of the expected masses in the three chromatographic peaks.
Figure 10. Pie chart of NIST mAb sub-unit protein across chromatographic peak: LC peak components pie chart (Left: LC peak 1; Right: LC peak 2).
Top-down (or middle-down) analyses have recently emerged as attractive alternatives to more traditional bottom-up approaches. However, they are not yet used routinely in analytical chemistry laboratories, partly because of the lack of software solutions.
This application note describes how the Intact Mass workflow in Byos can be used to automatically annotate MS/MS spectra in addition to deconvolution and mass matching of MS data.
The information generated can be used to quickly inform development teams and help investigate discrepancies in the different assays. Furthermore, as the visualization is easy for non-specialists to understand, it is also information that can be acted on more easily.
- J Proteome Res. 2018 Mar 2; 17(3): 1216–1226; doi: 10.1021/acs.jproteome.7b00839
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2.Analyst. 2016 May 23;141(11):3114-25. doi: 10.1039/c6an00071a
Rapid and improved characterization of therapeutic antibodies and antibody related products using IdeS digestion and subunit analysis
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