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

Abstract

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.

Precise quantitation of 136 urinary proteins by LC/MRM-MS using stable isotope labeled peptides as internal standards for biomarker discovery and/or verification studies

“…Furthermore, as evidenced by our quantified panel (see Supplemental Table 2), the less complex urine matrix enabled lower-abundance plasma proteins (e.g., macrophage colony-stimulating factor 1, P09603 [48]; osteopontin, P10451 [49]; protein DJ-1, Q99497 [50]), to be detected without multidimensional LC fractionation [51] or immunoaffinity-based depletion [52]. The cause is attributed to the lower concentration range of proteins in urine (approximately 6 orders of magnitude [53,54] vs. >10 in plasma [55]), which allows the high abundance plasma proteins to be quantified at significantly lower levels in urine. In fact, the average concentration of 9 typically depleted proteins in urine was found here to be 832 ng/mL compared to 4.2 mg/mL with a recently measured plasma sample [39] based on a matching set of quantifier peptides.…”

Similar Neutrophil-Driven Inflammatory and Antibacterial Responses in Elderly Patients with Symptomatic and Asymptomatic Bacteriuria

“…The urine sediment fraction, also termed the urinary pellet (UP), was resuspended with circa 10 ml phosphate-buffered saline (PBS), centrifuged, and resuspended in 200 to 400 l PBS before storage at Ϫ80°C until further use. Aliquots of the UP samples were thawed and subjected to denaturation and lysis using a solution containing 8 M urea, 50 mM dithiothreitol (DTT), 5 mM EDTA, and 1% SDS (vol/vol) and sonication followed by filter-aided sample preparation (FASP) in Vivacon filter devices with a 10-molecular-weight cutoff (MWCO) (Sartorius AG, Germany) as previously reported (25). Quantities of a lysate with approximately 10 to 100 g total urinary protein (estimated from Coomassie brilliant blue-G250 staining intensities of protein bands in SDS-PAGE gels) were digested twice with trypsin (25).…”

“…Second, UP samples have significant quantities of insoluble protein, leading to underestimation of protein content when colorimetric assays are used. The filtrate of the FASP process, enriched for peptides, was desalted using the StageTip method (26), and approximately one-fifth of the desalted filtrate was analyzed by liquid chromatography-tandem MS (LC-MS/MS) using a previously reported method (25). Briefly, an Ultimate 3000-nano LC workstation and a QExactive mass spectrometer system coupled with a FLEX nano-electrospray ion source (all components from Thermo Scientific, USA) were used.…”

MStern Blotting–High Throughput Polyvinylidene Fluoride (PVDF) Membrane-Based Proteomic Sample Preparation for 96-Well Plates

“…Although the application of FASP in the 96-well plate format has been described (3,4), the major limitation of FASP in the 96-well plate is the much slower speed at which the 96-well plates have to be centrifuged: while a single ultrafiltration unit withstands up to 14,000 ϫ g, the 96-well plate format can only be centrifuged at g-forces of up to 2,200 ϫ g. This significantly lower g-force for 96-well plates results in a slow liquid transfer, which in turn considerably prolongs the required centrifugation times to hours instead of tens of minutes for the three to four necessary centrifugation steps (i) for the initial loading, reduction and alkylation, (ii) for the different washing steps, and (iii) for the elution (3).…”