Expanding Proteome Coverage with CHarge Ordered Parallel Ion aNalysis (CHOPIN) Combined with Broad Specificity Proteolysis

The
“deep” proteome has been accessible by mass spectrometry
for some time. However, the number of proteins identified in cells
of the same type has plateaued at ∼8000–10 000
without ID transfer from reference proteomes/data. Moreover, limited
sequence coverage hampers the discrimination of protein isoforms when
using trypsin as standard protease. Multienzyme approaches appear
to improve sequence coverage and subsequent isoform discrimination.
Here we expanded proteome and protein sequence coverage in MCF-7 breast
cancer cells to an as yet unmatched depth by employing a workflow
that addresses current limitations in deep proteome analysis in multiple
stages: We used (i) gel-aided sample preparation (GASP) and combined
trypsin/elastase digests to increase peptide orthogonality, (ii) concatenated
high-pH prefractionation, and (iii) CHarge Ordered Parallel Ion aNalysis
(CHOPIN), available on an Orbitrap Fusion (Lumos) mass spectrometer,
to achieve 57% median protein sequence coverage in 13 728 protein
groups (8949 Unigene IDs) in a single cell line. CHOPIN allows the
use of both detectors in the Orbitrap on predefined precursor types
that optimizes parallel ion processing, leading to the identification
of a total of 179 549 unique peptides covering the deep proteome
in unprecedented detail.