For researchers in immunopeptidomics, the ability to capture a complete picture of the peptides presented by MHC molecules is central to understanding immune function and advancing therapeutic design. These peptides determine how the immune system distinguishes self from non-self and, as such, lie at the heart of many cutting-edge applications: vaccine development, cancer immunotherapy, autoimmune disease research, and more. Yet, despite its promise, immunopeptidomics remains one of the most technically challenging applications of mass spectrometry.
Until recently, most studies have relied on Data-Dependent Acquisition (DDA) or, more recently, DDA-library–based Data-Independent Acquisition (DIA). Both strategies have clear limitations. Semi-stochastic nature of DDA approach and its tendency to bias toward abundant peptides, leads to incomplete peptidome coverage, while library-based DIA requires significant effort to generate DDA-derived, comprehensive libraries. This is an extra step that requires larger sample amounts and increases measurement time, slowing down discovery. These approaches were a necessary compromise, given the complexity of immunopeptidome samples and their vast search spaces, as immunopeptides are characterized with unspecific digestion.
With Spectronaut® 20, we set out to change this landscape. Our goal was straightforward but ambitious: deliver unprecedented depth and speed in immunopeptide profiling, without the need to build a spectral library. At the core of this advance is Kuiper, a new search engine purpose-built for the unique challenges of immunopeptidomics. Kuiper utilizes a novel MS2 indexing concept, achieving fast search process with a small memory requirements. While working with Pulsar search engine, it benefits from new scores and improved deep learning models for unspecific searches accelerating searches while expanding peptidome coverage. The outcome: up to 80% more HLA type I and 30% more HLA type II peptide identifications compared to directDIA in Spectronaut 19, and dramatically faster directDIA analysis (up to 80%), empowering researchers to capture low-abundance, biologically critical peptides with confidence (n= 22 HLA type I and 4 HLA type II datasets). Spectronaut 20, powered by Kuiper, takes immunopeptidome analysis to the next level making analysis deeper, faster, and more reliable. Read on to see how these innovations reshape what is possible in immunopeptide research.
Spectronaut 20: Unlocking DIA Immunopeptidomics with directDIA
To realize the full potential of DIA-MS in immunopeptidomics, we needed to rethink completely our approach to meet the challenge of large search spaces. That’s why we developed Kuiper, a search engine designed specifically to handle the challenges of unspecific peptide searches. Unlike conventional tools that were adapted from proteomics workflows, Kuiper was built with unspecific peptide searches in mind and optimized for the complexity of large search spaces like those of HLA-bound peptides.
The result is a workflow that redefines immunopeptidome analysis. Kuiper enables faster searches, delivers deeper coverage, and maintains robust performance even in library-free mode. This makes Spectronaut 20 not just an incremental improvement, but a step change for researchers looking to push the boundaries of immunopeptide profiling.
Figure 1. HLA class I immunopeptide identifications, identifications classified as good binders marked with darker color.
Figure 2. Overlap of library-free and library-based identifications classified as good binders.
Here we look at a dataset from Wahle et al.1 which was not included in the training data, and we see Spectronaut 20’s library-free directDIA workflow significantly improves HLA class I peptide identifications compared with library-based analysis (Figure 1.). To demonstrate that the library free results are comparable to traditional library-based approach we plotted the overlap of peptides identified by each method (Figure 2.). There is a strong overlap between approaches: directDIA covers 85% of the good binding peptides found with a library-based workflow while also identifying 48% more peptides overall. By removing the constraints of pre-built libraries, Kuiper enables a broader exploration of the immunopeptidome.
Quality and Biological Relevance
In order to validate the directDIA approach for immunopeptidomics, we wanted to see if the high number of identified peptides also exhibit typical characteristics of HLA peptides.
Figure 3. HLA class I peptide length and charge distribution.
As expected, we observed strong agreement between workflows in key characteristics of immunopeptides such as charge states and peptide length distribution. DirectDIA reproduces hallmark features of HLA class I presentation, including a significant fraction of singly charged peptides and enrichment for 9-mers. These distributions confirm that the additional peptides identified are not artifacts but biologically meaningful.
Motif analysis further validates this conclusion.
Figure 4. Comparison of peptide motifs.
Peptide motifs capture the anchor residues that define antigen binding specificity for different HLA alleles. In immunopeptidomics, these motifs are critical because they directly connect peptide data to immune recognition. As shown in Figure 4, motifs derived from library-free directDIA are nearly identical to those from library-based analysis. This close match demonstrates that Kuiper not only expands coverage but does so while preserving biological fidelity.
Beyond Library-Based: A New Standard for Immunopeptidomics
Taken together, these results mark a significant advance for immunopeptidomics. For workflows where unspecific digestion and large search spaces make library generation difficult, library-free directDIA powered by Kuiper can not only match but outperform library-based approaches for certain experiments. At the same time, Spectronaut remains flexible: researchers who require libraries for their experimental designs can still rely on its industry-leading library-based analysis.
Beyond raw identifications, Spectronaut 20 also includes critical improvements to ensure reliability. We enhanced false discovery rate (FDR) control, giving researchers confidence in their peptide identifications. We also introduced new visualizations, such as peptide motifs, charge distributions, and length profiles that provide at-a-glance validation of results. These tools make it easier than ever to assess data quality, an essential step in any immunopeptidomics study.
Taking the Next Step
With its purpose-built Kuiper search engine and powerful directDIA workflow, Spectronaut 20 redefines what is possible in library-free immunopeptidomics analysis. Researchers can now achieve deeper coverage, faster results, and a more reliable view of the immunopeptidome all without the need for restrictive libraries.
The implications extend far beyond the lab. By unlocking more comprehensive datasets, Spectronaut 20 empowers discoveries that feed directly into translational applications, from designing next-generation vaccines to advancing cancer immunotherapy and understanding autoimmune disease. In a field where every peptide could point to a new therapeutic target, the ability to confidently detect more low-abundance peptides is transformative.
To learn more watch this seminar, which highlights how researchers are leveraging Spectronaut 20 to achieve that deeper, more comprehensive view of the immunopeptide landscape.
Spectronaut 20 raises the bar for immunopeptidome analysis. With Kuiper and directDIA, researchers can finally overcome the limitations of traditional methods and move toward a future where immunopeptidomics delivers its full potential.
Ready to take your research to the next level? Explore the possibilities with Spectronaut today.
References:
Wahle M, Thielert M, Zwiebel M, Skowronek P, Zeng WF, Mann M. IMBAS-MS Discovers Organ-Specific HLA Peptide Patterns in Plasma. Mol Cell Proteomics. 2024 Jan;23(1):100689. doi: 10.1016/j.mcpro.2023.100689. Epub 2023 Dec 1. PMID: 38043703; PMCID: PMC10765297.
