To support more sensitive immunopeptidomics analyses, Manakongtreecheep and Ctortecka et al. developed Pepyrus to rapidly and scalably generate user-defined peptide libraries in E. coli that could provide chromatographic and spectral references. Papyrus-enabled data-independent acquisition (DIA) allowed sensitive and high-confidence detection of lower-affinity and less abundant clinically relevant HLA-bound peptides, including neoantigens in complex patient samples. Further, a spectral library encompassing HIF-inducible ERVs for a renal cell carcinoma sample was reused “off-the-shelf” to analyze a melanoma sample, and identified ERV-derived peptides.
Contributed by Ute Burkhardt
ABSTRACT: Human leukocyte antigen (HLA)-bound tumor peptides can be routinely isolated from cancer samples and identified using mass spectrometry (MS). However, MS approaches can be stochastic or rely on spectral libraries, which are not customarily available for individual-specific peptides, thus limiting the ability to discover novel peptides. Here, we introduce Pepyrus, which generates user-defined, individual-specific or disease-specific peptide libraries in Escherichia coli to improve the sensitivity and confidence of MS peptide identification, including lowly abundant neoantigens. Using Pepyrus-generated peptide libraries paired with an HLA-specific data-independent acquisition strategy, we recover >75% of the expected sequences per single injection for libraries of >10,000 peptides and identify 0.1_fmol of spiked-in peptides in a complex background. We apply Pepyrus to create personalized libraries, facilitating identification of clinically relevant HLA peptides, including several novel peptides from cell lines derived from persons with melanoma and renal cell carcinoma. Pepyrus enables identification of rare HLA-bound peptides and provides the ability to generate large training datasets to improve spectra, retention time and ion mobility prediction tools.
Author Info: (1) Broad Institute of MIT and Harvard, Cambridge, MA, USA. Harvard Medical School, Boston, MA, USA. Dana Farber Cancer Institute, Boston, MA, USA. (2) Broad Institute of MIT and H
Author Info: (1) Broad Institute of MIT and Harvard, Cambridge, MA, USA. Harvard Medical School, Boston, MA, USA. Dana Farber Cancer Institute, Boston, MA, USA. (2) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (3) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (4) Broad Institute of MIT and Harvard, Cambridge, MA, USA. Dana Farber Cancer Institute, Boston, MA, USA. (5) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (6) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (7) Dana Farber Cancer Institute, Boston, MA, USA. (8) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (9) Dana Farber Cancer Institute, Boston, MA, USA. (10) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (11) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (12) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (13) Harvard Medical School, Boston, MA, USA. Dana Farber Cancer Institute, Boston, MA, USA. (14) Dana Farber Cancer Institute, Boston, MA, USA. (15) Department of Pathology, University of Michigan, Ann Arbor, MI, USA. (16) Dana Farber Cancer Institute, Boston, MA, USA. (17) Broad Institute of MIT and Harvard, Cambridge, MA, USA. Harvard Medical School, Boston, MA, USA. Dana Farber Cancer Institute, Boston, MA, USA. Department of Computer Science, Metropolitan College, Boston University, Boston, MA, USA. Technical University of Denmark, Lyngby, Denmark. (18) Broad Institute of MIT and Harvard, Cambridge, MA, USA. Harvard Medical School, Boston, MA, USA. Dana Farber Cancer Institute, Boston, MA, USA. (19) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (20) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (21) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (22) Broad Institute of MIT and Harvard, Cambridge, MA, USA. nhacohen@broadinstitute.org. Harvard Medical School, Boston, MA, USA. nhacohen@broadinstitute.org. Massachusetts General Hospital, Krantz Family Center for Cancer Research, Boston, MA, USA. nhacohen@broadinstitute.org. (23) Broad Institute of MIT and Harvard, Cambridge, MA, USA. scarr@broad.mit.edu. (24) Broad Institute of MIT and Harvard, Cambridge, MA, USA. jabelin@broadinstitute.org. Dana Farber Cancer Institute, Boston, MA, USA. jabelin@broadinstitute.org. (25) Broad Institute of MIT and Harvard, Cambridge, MA, USA. catherine_wu@dfci.harvard.edu. Harvard Medical School, Boston, MA, USA. catherine_wu@dfci.harvard.edu. Dana Farber Cancer Institute, Boston, MA, USA. catherine_wu@dfci.harvard.edu.
