Software-Assisted Data Processing Workflow for Intact Glycoprotein Mass Spectrometry

Intact protein analysis by mass spectrometry is important
for several
applications such as assessing post-translational modifications and
biotransformation. In particular, intact protein analysis allows the
detection of proteoforms that are commonly missed by other approaches
such as proteolytic digestion followed by bottom-up analysis. Two
quantification methods are mainly used for intact protein data quantification,
namely the extracted ion and deconvolution approaches. However, a
consensus with regard to a single best practice for intact protein
data processing is lacking. Furthermore, many data processing tools
are not fit-for-purpose and, as a result, the analysis of intact proteins
is laborious and lacks the throughput required to be implemented for
the analysis of clinical cohorts. Therefore, in this study, we investigated
the application of a software-assisted data analysis and processing
workflow in order to streamline intact protein integration, annotation,
and quantification via deconvolution. In addition, the assessment
of orthogonal data sets generated via middle-up and bottom-up analysis
enabled the cross-validation of cleavage proteoform assignments present
in seminal prostate-specific antigen (PSA). Furthermore, deconvolution
quantification of PSA from patients’ urine revealed results
that were comparable with manually performed quantification based
on extracted ion electropherograms. Overall, the presented workflow
allows fast and efficient processing of intact protein data. The raw
data is available on MassIVE using the identifier MSV000086699.