Urine sample centrifugation and concentration. A human subject urine specimen obtained from a clinical laboratory usually has a volume in the range of 5–50 mL. The ideal workflow starts with the processing of urine soon after collection, without intermittent freezing. A urine sample can be kept at 4 °C for up to 10 h prior to the centrifugation; however, salts may precipitate during cold storage. If it is not possible to process a urine specimen within 10 h, it can be frozen at −20 °C or −80 °C at the clinical site and shipped to the analytical laboratory when convenient. The freshly collected or thawed urine sample is equilibrated to 20 °C and neutralized to a pH of 7–8 with a 1 M Tris-HCl solution (pH 8). Urine tends to have a slightly acidic pH (6–7), but can occasionally be more acidic or basic. Neutralization at 20 °C usually dissolves crystallized uric acid and calcium oxalate, sodium urate, and magnesium ammonium phosphate salts if present.

Centrifuge the urine sample in a conical tube at 3,000 rpm for 15 min at 10–15 °C, carefully collect the urine supernatant, transfer to a new tube, and retain for further processing of the urine supernatant. Also recover the pellet retaining 0.5–1 mL of urine supernatant to avoid disturbing the urinary sediment (pellet).

Urine pellet: add ~10 mL phosphate-buffered saline (PBS) at 4 °C, gently shake the tube to resuspend the pellet and centrifuge for 15 min at 3,000 rpm. Discard this supernatant (the wash solution) and store urinary pellet at −80 °C or process immediately.

Urine supernatant: to concentrate the urine supernatant, transfer it to an Amicon Ultra-15 filter, and concentrate at 3,000 rpm to a volume of ~1.0 mL for 15–30 min. Multiple consecutive concentration steps may be needed if supernatant volume is larger than 15 mL and if the filtration rate is low. Wash the urine supernatant concentrate with ~10 mL PBS and spin again to reduce the volume to ~1.0 mL.

Urine supernatant: the total protein quantity in the urine concentrate is measured using Bradford Protein Assay (BioRad). To support the quantitative estimate and assess the relative abundance of albumin in the concentrate, an SDS-PAGE gel can be run.

Urine supernatant: aliquot 20–100 μg of the urinary concentrate, mix with 1 % SDS and 10 mM DTT (final concentration) and boil in microcentrifuge tube for 10–15 min at 95 °C. Cool to room temperature and the samples are ready for the 96FASP method.

Urine pellet: independent of the cellular composition of the urinary pellet, a cell disruption and protein solubilization step is required to maximize the number of distinct proteins effectively digested during the FASP analysis and to estimate the amount of total protein to be subjected to FASP and digested with a proteolytic enzyme (trypsin). To achieve effective lysis of most cells in a urinary pellet, add USED buffer with an approximate volume ratio of 4:1 (buffer/urinary pellet). If the urinary pellet volume is very small, a minimum of 100 µL USED buffer volume should be added. Resuspend by vortexing vigorously (2 or 3 times for 30 s).

Urine pellet: pretreatment with lysostaphin and mutanolysin. Many Gram-positive bacteria have thick cell walls (e.g., various Streptococcus and Staphylococcus species). USED buffer suspension followed by sonication is not effective in lysing these cells. If the purpose of a urine pellet proteomic analysis is to investigate for UTI, possibly caused by Streptococcus or Staphylococcus species, resuspend the urinary pellet in TMN buffer in a volume ratio of 1:10 (pellet/TMN), usually less than 200 µL. Pipette the suspension up and down a few times in the microcentrifuge tube. Add lysostaphin and mutanolysin each to a final concentration of 20 µg/mL. Mix gently to homogenize enzymes and suspended cells. Incubate sample in a shaker-incubator at 37 °C for 60–150 min, while occasionally mixing to resuspend cells/cellular debris.

Note In the case that a urinary pellet is pretreated with lysostaphin and mutanolysin, add up to 600 µL USED buffer to the lysate. Resuspend by vortexing vigorously (2 or 3 times for 30 s).

Urine pellet: using Misonex Sonicator with the water bath hooked up to it (not the probe), place urinary pellet lysates in microcentrifuge tube (from steps 7 and/or 8) in a float in the water bath filled with ice water. Set the program at amplitude 9 and sonicate for nine 45-s “on” cycles and 45-s intermittent “off” cooling cycles. The cells in a urinary pellet are disrupted further.

Urine pellet: let urinary lysate sit for approximately 5 min and spin in a bench top microcentrifuge at maximum speed (13,000 rpm) for 10 min. Transfer the lysate supernatant into a new 1.5 mL microtube. Discard pellet.

Urine pellet: take a 10 µL aliquot of the urinary pellet lysate (supernatant), mix with SDS-loading buffer (for SDS-PAGE gels) and run lysate(s) in a SDS-PAGE gel. Freeze the remaining lysate(s) at −80 °C until further use.

Urine pellet: load 2 and 4 µg BSA standards in the same gel. Coomassie Blue (CB)-G250 stain the gel followed by destaining with standard procedures [16]. From the overall CB-G250 staining intensity of urinary pellet lysate bands, estimate total protein amount per lane (lysate) compared to BSA band staining intensity.

Load approximately 10–50 µg total protein (from urine supernatant concentrate or the urinary pellet lysate) into the 96FASP filter plate, and mix with 200 µL Urea buffer.

Spin at 3,600 rpm for around 45 min.

Note Different brands of centrifuges may perform differently even at the same spin centrifugal force. In the hands of our laboratory, the Eppendorf 5810R centrifuge worked better than the Beckman Coulter centrifuge (Allegra 6R).

Add 200 µL urea buffer and repeat the spin at 3,600 rpm to concentrate until the volume in the well is reduced to ~10 µL.

Add 100 µL of IAA solution (final concentration 50 mM). Incubate for 20 min in the dark at room temperature, and spin for ~45 min.

Add 200 µL of urea buffer and spin again. The final sample volume in the filter unit should be 20 µL or less.

Add 200 µL of ABC buffer and spin. Repeat this step one time.

Add 100 µL of ABC buffer and then add trypsin in a ratio 1:50 (trypsin–protein). Mix gently and incubate overnight at 37 °C.