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  • Brief Communication
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A learned embedding for efficient joint analysis of millions of mass spectra

Nature Methods volume 19, pages 675–678 (2022)Cite this article

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Abstract

Computational methods that aim to exploit publicly available mass spectrometry repositories rely primarily on unsupervised clustering of spectra. Here we trained a deep neural network in a supervised fashion on the basis of previous assignments of peptides to spectra. The network, called ‘GLEAMS’, learns to embed spectra in a low-dimensional space in which spectra generated by the same peptide are close to one another. We applied GLEAMS for large-scale spectrum clustering, detecting groups of unidentified, proximal spectra representing the same peptide. We used these clusters to explore the dark proteome of repeatedly observed yet consistently unidentified mass spectra.

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Fig. 1: GLEAMS deep neural network architecture and embedding performance.
Fig. 2: Exploration of the dark proteome using GLEAMS to process previously unidentified spectra.

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Data availability

The data used to explore the dark proteome have been deposited to the MassIVE repository with the dataset identifier MSV000088598. It consists of MGF files containing the representative medoid spectra from GLEAMS clustering and the associated ANN-SoLo identifications in mzTab format27.

All other data supporting the presented analyses have been deposited to the MassIVE repository with the dataset identifier MSV000088599. Source data are provided with this paper.

Code availability

GLEAMS was implemented in Python 3.8. Pyteomics (v.4.3.2)45 was used to read MS/MS spectra in the mzML22, mzXML and MGF formats. spectrum_utils (v.0.3.4)46 was used for spectrum preprocessing. We performed a submodular selection using apricot (v.0.4.1)21. The neural network code was implemented using the Tensorflow/Keras framework (v.2.2.0)47. SciPy (v.1.5.0)48 and fastcluster (v.1.1.28)49 were used for hierarchical clustering. Additional scientific computing was done using NumPy (v.1.19.0)50, Scikit-Learn (v.0.23.1)51, Numba (v.0.50.1)52 and Pandas (v.1.0.5)53. Data analysis and visualization were performed using Jupyter Notebooks54, matplotlib (v.3.3.0)55, Seaborn (v.0.11.0)56 and UMAP (v.0.4.6)13.

All code is available as open source under the permissive BSD license at https://github.com/bittremieux/GLEAMS. Code used to analyze the data and to generate the figures presented here is available on GitHub (https://github.com/bittremieux/GLEAMS_notebooks). Permanent archives of the source code and the analysis notebooks are available on Zenodo at https://doi.org/10.5281/zenodo.5794613 and https://doi.org/10.5281/zenodo.5794616, respectively57,58.

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Acknowledgements

This work was supported by National Institutes of Health award R01 GM121818.

Author information

Authors and Affiliations

  1. Skaggs School of Pharmacy and Pharmaceutical Science, University of California San Diego, La Jolla, CA, USA

    Wout Bittremieux

  2. Department of Genome Sciences, University of Washington, Seattle, WA, USA

    Damon H. May & William Stafford Noble

  3. Department of Electrical and Computer Engineering, University of Washington, Seattle, WA, USA

    Jeffrey Bilmes

  4. Paul G. Allen School of Computer Science and Engineering, University of Washington, Seattle, WA, USA

    Jeffrey Bilmes & William Stafford Noble

Authors
  1. Wout Bittremieux
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Contributions

W.S.N. conceptualized the work. W.S.N. and J.B. supervised the work. W.B. and D.H.M. developed the software and carried out the analyses. W.B., D.H.M. and W.S.N. wrote the manuscript. All authors reviewed and edited the manuscript.

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Correspondence to William Stafford Noble.

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Peer review information

Nature Methods thanks Johannes Griss, Sean Hackett and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Allison Doerr, in collaboration with the Nature Methods team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 GLEAMS embedder network.

Each instance of the embedder network in the Siamese neural network separately receives each of three feature types as input. Precursor features are processed through a fully connected network with two layers of sizes 32 and 5. Binned fragment intensities are processed through five blocks of one-dimensional convolutional layers and max pooling layers. Reference spectra features are processed through a fully connected network with two layers of sizes 750 and 250. The output of the three subnetworks is concatenated and passed to a final fully connected layer of size 32.

Extended Data Fig. 2 UMAP visualization of embeddings, colored by precursor charge.

UMAP projection of 685,337 embeddings from frequently occurring peptides in 10 million randomly selected identified spectra. Note that the visualization may group peptides with similarities on some dimensions of the 32-dimensional embedding space, but which are nevertheless distinguishable based on their full embeddings.

Source data

Extended Data Fig. 3 False negative rate between positive and negative embedding pairs.

The false negative rate between positive and negative embedding pairs for 10 million randomly selected pairs from the test dataset, at distance threshold 0.5455 (gray line), corresponding to 1% false discovery rate, is 1%.

Source data

Extended Data Fig. 4 ROC curve for GLEAMS performance on unseen phosphorylated spectra.

Receiver operating characteristic (ROC) curve for GLEAMS embeddings corresponding to 7.5 million randomly selected spectrum pairs from an independent phosphoproteomics study. The ROC curve and area under the curve (AUC) show how often a same-peptide spectrum pair had a smaller distance than a different peptide spectrum pair.

Source data

Extended Data Fig. 5 Clustering result characteristics produced by different tools.

Clustering result characteristics at approximately 1% incorrectly clustered spectra over three random folds of the test dataset. (a) Complementary empirical cumulative distribution of the cluster sizes. (b) The number of datasets that spectra in the test dataset originate from per cluster (24 datasets total).

Source data

Extended Data Fig. 6 GLEAMS performance with different clustering algorithms.

Average clustering performance over three random folds of the test dataset containing 28 million MS/MS spectra each. The GLEAMS embeddings were clustered using hierarchical clustering with complete linkage, single linkage, or average linkage; or using DBSCAN. The performance of alternative spectrum clustering tools (Fig. 1d, e) is shown in gray for reference. (a) The number of clustered spectra versus the number of incorrectly clustered spectra per clustering algorithm. (b) Cluster completeness versus the number of incorrectly clustered spectra per clustering algorithm.

Source data

Extended Data Fig. 7

Runtime scalability of spectrum clustering tools. Scalability of spectrum clustering tools when processing increasingly large data volumes. Three random subsets of the test dataset were combined to form input datasets consisting of 28 million, 56 million, and 84 million spectra. Evaluations of falcon and MS-Cluster on larger datasets were excluded due to excessive runtimes.

Source data

Extended Data Fig. 8 UMAP visualization of the selected reference spectra.

UMAP visualization of the selected reference spectra. The two-dimensional UMAP visualization was computed from the dot product pairwise similarity matrix between all 200,000 randomly selected spectra from the training data.

Source data

Extended Data Fig. 9 Input features ablation test.

Ablation testing during training of the GLEAMS Siamese network shows the benefit of the different input feature types. The performance is measured using the validation loss while training for 20 iterations consisting of 40,000 steps with batch size 256. The line indicates the smoothed average validation loss over five consecutive iterations, with the markers showing the individual validation losses at the end of each iteration.

Source data

Supplementary information

Reporting Summary

Peer Review File

Supplementary Table 1

GLEAMS learns latent spectrum properties. Correlation of individual embedding dimensions with latent properties of the spectra. Spearman correlations above 0.2 and below −0.2 are shown.

Supplementary Table 2

Top 500 precursor mass differences from the ANN-SoLo open modification search of GLEAMS cluster centroids.

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Bittremieux, W., May, D.H., Bilmes, J. et al. A learned embedding for efficient joint analysis of millions of mass spectra. Nat Methods 19, 675–678 (2022). https://doi.org/10.1038/s41592-022-01496-1

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