Cysteine Counting via Isotopic Chemical Labeling for Intact Mass Proteoform Identifications in Tissue

Top-down proteomics, the tandem mass spectrometric analysis
of
intact proteoforms, is the dominant method for proteoform characterization
in complex mixtures. While this strategy produces detailed molecular
information, it also requires extensive instrument time per mass spectrum
obtained and thus compromises the depth of proteoform coverage that
is accessible on liquid chromatography time scales. Such a top-down
analysis is necessary for making original proteoform identifications,
but once a proteoform has been confidently identified, the extensive
characterization it provides may no longer be required for a subsequent
identification of the same proteoform. We present a strategy to identify
proteoforms in tissue samples on the basis of the combination of an
intact mass determination with a measured count of the number of cysteine
residues present in each proteoform. We developed and characterized
a cysteine tagging chemistry suitable for the efficient and specific
labeling of cysteine residues within intact proteoforms and for providing
a count of the cysteine amino acids present. On simple protein mixtures,
the tagging chemistry yields greater than 98% labeling of all cysteine
residues, with a labeling specificity of greater than 95%. Similar
results are observed on more complex samples. In a proof-of-principle
study, proteoforms present in a human prostate tumor biopsy were characterized.
Observed proteoforms, each characterized by an intact mass and a cysteine
count, were grouped into proteoform families (groups of proteoforms
originating from the same gene). We observed 2190 unique experimental
proteoforms, 703 of which were grouped into 275 proteoform families.