Calibration std conc. (pg/mL)
Add working calibration std Mix 1, 1 pg/μL (μL)
Add working calibration std Mix 2, 10 pg/μL (μL)
Add working calibration std Mix 3, 100 pg/μL (μL)
Add serum PP depleted (μL)
30
30
450
50
5
450
100
10
450
200
20
450
500
5
450
1000
10
450
8.
Quality control (QC) samples: This assay uses three controls along the run to confirm assay accuracy. One negative control and two controls with low and high peptides levels are analyzed with every batch of samples. The QC samples are prepared by spiking PP and PP3-36 into patient serum pools.
(a)
Negative control: pooled PP and PP3-36 depleted serum/plasma .
(b)
Control level 1 (low): 30 pg/mL PP and PP3-36. 50 mL of pooled, depleted serum is spiked with 15 μL of 100 pg/μL of PP and PP3-36 (Mixture 3). Aliquot 500 μL in 1.5 mL microcentrifuge LoBind vials and store at −70 °C. Stable for 6 months.
(c)
Control level 2 (high): 500 pg/mL PP and PP3-36. 50 mL of pooled, depleted serum is spiked with 20 μL of 1250 pg/μL of target peptides (Mixture 4). Aliquot 500 μL in 1.5 mL microcentrifuge LoBind™ tubes and store at −70 °C. Stable for 6 months.
2.4 Equipment
1.
Triple quadrupole mass spectrometer API 5500 (ABSciex) with TurboV ion source. Software Analyst 1.6.1 or newer version.
2.
Binary HPLC pumps series 1200 SL (Agilent Technologies), vacuum degasser, autosampler CTC-PAL (LEAP Technologies) equipped with fast wash station.
3.
Centrifuge.
4.
Sample rocker mixer for 1.5 mL microcentrifuge tubes.
5.
Microcentrifuge for 0.5 and 1.5 mL tubes.
6.
Analytical balance.
2.5 Supplies
1.
1.5 mL microcentrifuge LoBind™ tubes (Eppendorf).
2.
Polypropylene 0.3 mL vials (Wheaton, Millville, NJ).
3.
PTFE/rubber cap (National Scientific, Rockwood, TN).
4.
Filter Units 500 mL, 0.45 μm nylon membrane (Nalgene, Rochester, NY).
5.
Glass funnel filter with vacuum adapter. Sigma-Aldrich.
6.
Guard cartridge Poroshell 300SB-C18 with Guard Column cartridge holder (Agilent Technologies, Santa Clara, CA).
7.
HPLC column for analytical separation Poroshell 300SBC18 column 2.1 × 75 mm, 5 μm (Agilent Technologies).
3 Methods
3.1 Procedure for Sample Preparation (See Note 7 )
2.
450 μL of patient samples and controls are aliquoted in 1.5 mL microcentrifuge protein LoBind™ vials.
3.
Add to each tube 225 μL of Trizma buffer 3×.
4.
Add 10 μL of working internal standard.
5.
Resuspend magnetic beads and add 5 μL to each tube.
6.
Incubate for 1 h at 4 °C. Ensure beads are suspended by gentle vortexing.
7.
Remove supernatant. Set tubes on magnet stand and remove supernatants with a pipette, changing tips for each sample.
8.
Wash the beads twice with 500 μL Trizma 1×. Set tubes on magnet stand and remove supernatants with a pipette, changing tips for each sample. Mix samples between the washes.
9.
Wash the beads once with 500 μL Trizma 0.2× with deionized water (see Note 6 ). Set tubes on magnet stand and remove supernatants with a pipette, changing tips for each sample.
10.
Add 40 μL of 5 % acetic acid with 0.0025 % BSA to elute peptides. Incubate at room temperature for 10 min (without vortexing).
11.
Set tubes on magnet stand and transfer the supernatants to polypropylene 0.3 mL vials (do not transfer the beads). Seal the vials with PTFE/rubber caps. Centrifuge vials at 2000 × g for 30 s.
12.
Set the vials to the CTC-PAL autosampler for LC-MS/MS analysis.
3.2 HPLC and Autosampler CTC-PAL Conditions
1.
HPLC conditions are provided in Table 2. Column temperature is 70 °C.
Table 2
Mobile phase program for chromatographic separation
Step | Time | Flow rate (μL/min) | %A (H2O + 10 mM acetic acid, 5 % ACN) | %B (Acetonitrile + 10 mM acetic acid) |
|---|---|---|---|---|
1 | 0.00 | 700 | 95.0 | 5.0 |
2 | 0.50 | 700 | 79.0 | 21.0 |
3 | 2.00 | 700 | 42.0 | 58.0 |
4 | 2.01 | 700 | 5.0 | 95.0 |
5 | 4.00 | 700 | 5.0 | 95.0 |
6 | 4.01 | 700 | 95.0 | 5.0 |
7 | 4.50
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