Urine Sample Preparation in 96-Well Filter Plates for Quantitative Clinical Proteomics

Urine is an important, noninvasively
collected body fluid source
for the diagnosis and prognosis of human diseases. Liquid chromatography
mass spectrometry (LC-MS) based shotgun proteomics has evolved as
a sensitive and informative technique to discover candidate disease
biomarkers from urine specimens. Filter-aided sample preparation (FASP)
generates peptide samples from protein mixtures of cell lysate or
body fluid origin. Here, we describe a FASP method adapted to 96-well
filter plates, named 96FASP. Soluble urine concentrates containing
∼10 μg of total protein were processed by 96FASP and
LC-MS resulting in 700–900 protein identifications at a 1%
false discovery rate (FDR). The experimental repeatability, as assessed
by label-free quantification and Pearson correlation analysis for
shared proteins among replicates, was high (R ≥
0.97). Application to urinary pellet lysates which is of particular
interest in the context of urinary tract infection analysis was also
demonstrated. On average, 1700 proteins (±398) were identified
in five experiments. In a pilot study using 96FASP for analysis of
eight soluble urine samples, we demonstrated that protein profiles
of technical replicates invariably clustered; the protein profiles
for distinct urine donors were very different from each other. Robust,
highly parallel methods to generate peptide mixtures from urine and
other body fluids are critical to increase cost-effectiveness in clinical
proteomics projects. This 96FASP method has potential to become a
gold standard for high-throughput quantitative clinical proteomics.