Identification and Mitigation of Defensins in the Immunopurification of Peptide MHC‑I Antigens from Lung Tissue

The class I major histocompatibility (MHC-I) complex
is a set of
diverse cell surface receptors encoded by the human leukocyte antigen
gene complex. These receptors present intracellular antigens to cytotoxic
T cells providing information on the state and health of cells. Changes
in the immunopeptidome during cancer may provide novel targets for
therapeutic intervention. To understand how the tumor immunopeptidome
is altered, we developed a mass spectrometry (MS) based platform for
isolating and identifying MHC-I peptide antigens in lung tumors. In
the course of our work, we encountered several large unknown peptide
contaminants which had not been previously reported. To understand
the source of these major contaminants, we isolated them using offline
fractionation and identified them by liquid chromatography–tandem
mass spectrometry (LC–MS/MS) as members of the host defense
protein family known as the defensins. To mitigate their detrimental
effects, we modified our “Original” data-dependent acquisition
(DDA) MS method to narrowly target the MHC-I peptides based on their
physical properties including charge state and molecular weight (“z
state” DDA), evaluated field asymmetric ion mobility spectrometry
to attempt gas-phase separation prior to MS analysis, and developed
an immunodepletion approach using defensin specific antibodies. This
modified approach improves peptide identification and reduces the
impact of defensin contamination in lung tissue samples.