Advancing Urinary Protein Biomarker Discovery by Data-Independent Acquisition on a Quadrupole-Orbitrap Mass Spectrometer

The
promises of data-independent acquisition (DIA) strategies are
a comprehensive and reproducible digital qualitative and quantitative
record of the proteins present in a sample. We developed a fast and
robust DIA method for comprehensive mapping of the urinary proteome
that enables large scale urine proteomics studies. Compared to a data-dependent
acquisition (DDA) experiments, our DIA assay doubled the number of
identified peptides and proteins per sample at half the coefficients
of variation observed for DDA data (DIA = ∼8%; DDA = ∼16%).
We also tested different spectral libraries and their effects on overall
protein and peptide identifications and their reproducibilities, which
provided clear evidence that sample type-specific spectral libraries
are preferred for reliable data analysis. To show applicability for
biomarker discovery experiments, we analyzed a sample set of 87 urine
samples from children seen in the emergency department with abdominal
pain. The whole set was analyzed with high proteome coverage (∼1300
proteins/sample) in less than 4 days. The data set revealed excellent
biomarker candidates for ovarian cyst and urinary tract infection.
The improved throughput and quantitative performance of our optimized
DIA workflow allow for the efficient simultaneous discovery and verification
of biomarker candidates without the requirement for an early bias
toward selected proteins.