SUPPLEMENTAL MATERIALS AND METHODS

Transcription

1 Supplemental materials for manuscript Kushnir MM, Rockwood AL, Strathmann FG, Frank EF, Straseski JA, Meikle AW. High Sensitivity Measurement of Parathyroid Hormone Related Peptide Using LC-MS/MS SUPPLEMENTAL MATERIALS AND METHODS Reagents, standards Calibration standard of PTHrP (0.75 pg/µl) was prepared from recombinant PTHrP (AAS ; AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEIRATSEVSPNSKPSP NTKNHPVRFGSDDEGRYLTQETNKVETYKEQPLKTP) purchased from PeproTech (Rocky Hill, NJ)) in fetal bovine serum, lyophilized and stored frozen at 70 C. The working calibrators were prepared in 0.1% BSA at concentrations 1, 2, 5, 10 and 30 pmol/l. Rabbit polyclonal anti PTHrP antibody was purchased from PeproTech. Plasma quality control samples were pooled human plasma at concentrations of 4 and 11 pmol/l of PTHrP. The recombinant 15N labeled internal standard ( 15 N PTHrP) was diluted in BSA to a concentration of 1 nmol/l and frozen at 20 C until used. Trypsin (purity 99%, activity 15,000 BAEE units), Tris(hydroxymethyl)aminomethane (TRIS), (4 (2 hydroxyethyl) 1 piperazineethanesulfonic acid (HEPES), formic acid, and acetic acid were purchased from Sigma Aldrich (St. Louis, MO). All other reagents were of the highest purity commercially available. Solvents were of HPLC grade, purchased from JT Baker (Phillipsburg, NJ). The polyclonal rabbit anti PTHrP antibody (PeproTech) was conjugated to Tosyl activated magnetic beads (Life Technologies, Carlsbad, CA) according to the manufacturer s recommendations. Briefly, the beads were washed with buffer and re suspended in 1M ammonium sulfate solution containing 20 μg of the antibody per milligram of beads. Beads were incubated at 37 C for 20 h, then incubated for 1 h with blocking buffer, washed twice using the wash buffer and reconstituted to a concentration of 20 mg/ml using the storage buffer. The conjugation was performed in 1.5mL microcentrifuge tubes; 670µL aliquot of the beads was transferred in the tube and 400 ug of the anti peptide antibody was used for the conjugation; 1mL of the buffers was used for the bead washes and blocking; the final reconstitution volume was 960uL.

2 The blocking buffer (PBS with 0.5% BSA, ph 7.4) was prepared as follows: to 80 ml of 0.01M sodium phosphate (ph 7.4), 0.88 g of NaCl and 0.5g BSA were added, the volume was adjusted with deionized water to 100 ml; ph was adjusted to 7.4. The wash/ storage buffer was prepared as follows: to 80 ml of 0.01M sodium phosphate (ph 7.4), 0.88 g of NaCl and 0.1g BSA were added, the volume was adjusted with deionized water to 100 ml; ph was adjusted to 7.4. The bead washes were performed using magnetic stand for micerocentrifuge tubes. Sample preparation Sample preparation was performed on a liquid handler Janus (Perkin Elmer, CA). 400 μl of HEPES buffer (ph 7.4) and 20 μl of internals standard were added to a 400μL aliquot of plasma sample and the samples incubated for 15 min. After the incubation, 5 μl of the magnetic beads suspension was added and the samples were incubated with agitation at 10 C for 3 h. The beads were washed three times with TRIS buffer (ph 7.4), then 200 μl of 25 mm bicarbonate buffer and 10 μl of trypsin (4 μg/μl) were added and the samples incubated for 3 h at 37 C. After the digestion, 10 μl of 10% formic acid was added to the samples, the tubes were vortexed, the samples transferred into wells of a 96 well plate, and 70 μl aliquots injected into the LC MS/MS. Magnetic beads were processed using magnetic stand for 96 well plates. Effect of the conditions of the denaturing, reduction, alkylation, digestion, affinity enrichment, magnetic beads washes and peptide elution was evaluated to optimize method performance. Experiments showed no effect of the denaturing, cysteines reduction and alkylation on the recovery of the targeted peptide; therefore, reduction and alkylation were not used in this method. Instrumental analysis The two dimensional (2D) HPLC separation was performed on an HPLC system consisting of series 1260 and 1290 pumps (Agilent Technologies, Santa Clara, CA). A Synergy Polar RP, 50x3mm, 4 μm HPLC column (Phenomemex, Torrance, CA) was used for the first dimension separation with a gradient of 99% to 87% A in 2.7 min (A, 10 mm formic acid in water; B, 10 mm formic acid in acetonitrile); the second dimension

3 separation was on a Synergy Max RP, 100x3mm, 2.5μm, column (Phenomemex) using a gradient of 97% to 85% A in 2 min (A, 5 mm acetic acid in water; B, 5 mm acetic acid in acetonitrile). The LC separation was performed at 40 C. Quantitative analysis was performed on an AB5500 mass spectrometer (AB Sciex, Framingham, MA) with a V spray ionization source in a positive ion, multiple reaction monitoring mode. Mass transitions monitored were m/z and for the YLTQETNK peptide, and m/z and for the internals standard. The instrument settings were adjusted to maximize the sensitivity and the specificity of detection. The heating gas temperature was 650 C, the nebulizing, curtain and collision gases (nitrogen) were 60 psi, 60 psi and 9 arbitrary units. The declustering potential, collision energy, collision cell exit potential, and entrance potential were 60V, 23V, 26V, and 10V. The dwell time for the mass transitions was 40 ms. The Q1 and Q3 quadrupoles were set to unit resolution. The total analysis time per sample was 6.5 min. The instrument control and the data analysis were performed using Analyst (AB Sciex). Method validation Method validation consisted of the evaluation of the imprecision, determining limit of quantification (LOQ), limit of detection (LOD), linearity, comparison of the method with commercial RIA (Immunotech), recovery, carryover, ion suppression, and establishment of reference intervals. Plasma pools used during the method validation were prepared from remaining aliquots of patient samples submitted for PTHrP testing to ARUP laboratories. An assessment of intra assay and inter assay imprecision was performed by analyzing pools of human plasma samples containing endogenous PTHrP or supplemented with recombinant PTHrP (PeproTech). Concentrations of PTHrP in the samples were 4, 12, and 48 and 398 pmol/l. The samples were analyzed in three replicates over seven days. In addition, three quality control samples were analyzed in

4 routine runs over 20 d. All studies were approved by the Institutional Review Board of the University of Utah. Limits of detection and quantification were evaluated by analyzing plasma samples containing progressively lower concentration of PTHrP. Four samples were prepared by dilution of plasma pool containing 4.5 pmol/l with HEPES buffer; the samples were analyzed in triplicate over three days. Linearity of the method was evaluated by analyzing seven samples prepared by mixing in different proportions two plasma pools containing 8 and 580 pmol/l of PTHrP. The samples were analyzed in duplicate over two days. The limit of quantification and upper limit of linearity were determined as the lowest and the highest concentrations, respectively, at which accuracy was within ±15%, imprecision was <20% and a ratio of the mass transitions were maintained within ±30%. LOD was the lowest concentration at which chromatographic peaks were present in all mass transitions and signal to noise ratio was >7. Blank samples were injected after high standards to evaluate carryover potential of the method. The method was compared with PTHrP RIA (Immunotech); samples used for the evaluation were remaining aliquots of patient plasma samples submitted to ARUP laboratories for routine testing. Three sets of plasma samples were used for the method evaluation: 1) samples previously analyzed for PTHrP by the Immunotech RIA (n=207), 2) samples containing elevated calcium (>1.05 mg/l) and low PTH (<15 pg/ml; n=88), and 3) samples with normal calcium and low PTH (<15 pg/ml; n=44). Differences in concentrations among the groups were evaluated using Wilcoxon rank sum test. Qualitative confirmation of PTHrP was assessed through the ratio of the concentrations determined from two mass transitions of the targeted peptide and the internal standard (17); ratio of the mass transitions outside of the ±30% range or presence of split peaks was considered as evidence of interfering peaks. The affinity enrichment recovery was determined by performing enrichment of a plasma pool of patient samples containing 10 pmol/l of PTHrP. Internal standard was

5 added to three samples before, and to another three samples after the enrichment, and the rest of the sample preparation was performed as described above. The difference between the observed concentrations of PTHrP in the pre and post enrichment spiked samples gave a measure of the recovery. Sample dilution integrity was evaluated by analyzing patient plasma sample containing 180 pmol/l of PTHrP. The sample was analyzed (in duplicate) with dilutions 3x, 5x, 10x, 20x, and100x. The dilution was performed with a plasma sample containing 1.2 pmol/l of PTHrP. Effect of lipemia, hemolysis and icterus was evaluated by analyzing pools of normal plasma samples containing 5 pmol/l of PTHrP and the same pool spiked with intralipid (1.1%), bilirubin (150 mg/l), and hemolyzed red blood cells (1.5 g/l); concentrations observed in the samples were compared. Ion suppression was evaluated using post column infusion method (18). A set of patient samples containing low concentrations of PTHrP was analyzed by the method, while standard of the peptide YLTQETNK (100 ng/ml) was infused into the effluent of the analytical column. The chromatograms were inspected for signs of ion suppression. Blood collection tubes suitability was evaluated using six types of tubes with blood drawn from five volunteers. Blood was drawn in potassium EDTA, sodium heparin, lithium heparin, serum, serum separation, and Phe Pro Arg chloromethylketone inhibitor tubes (PPACK, Haematologic Technologies, Essex Junction, VT). PTHrP concentrations observed in the samples were compared within each individual; samples were analyzed fresh and after storage in refrigerator for 48h. Stability was also evaluated by repeat analysis of patient samples (collected in tubes containing PPACK inhibitor) processed on ice until aliquoted, then returned in 70 C freezer and defrosted for the repeat testing.

6 Reference interval study for PTHrP was performed with samples from self reported healthy adult volunteers, 114 men (ages 20 58, median 33), and 122 women (ages 20 67, median 32). Body mass index (BMI) of the participants was calculated as weight (kg) divided by height (m) squared. The volunteers were without chronic diseases; blood was collected in tubes with PPACK inhibitor, plasma was separated from blood cells within one hour, and the samples were stored at 70 C.

7 Supplemental Table 1. Within-run, between-run total imprecision. Imprecision, % Mean concentration, pmol/l within-run betweenrun/day total 4.3 (pool of patient samples)* (pools of patient samples spiked with recombinant PTHrP)* (pools of patient samples spiked with recombinant PTHrP)* (pools of patient samples analyzed as QC)** na 13.4 na 11.8(pools of patient samples analyzed as QC)** na 12.2 na * - data correspond to plasma samples containing PTHrP, analyzed in triplicate over 7 days. ** - data correspond to QC samples (plasma samples containing PTHrP) analyzed over 20 days in single replicate per day.

8 e5 6.0e5 Intensity, cps e m/z, Da Supplemental Figure 1. Product ion mass spectrum of the PTHrP specific peptide YLTQETNK.

9 3.5 PTHrP, pmol/l p= POSTMENOPAUSAL PREMENOPAUSAL Supplemental Figure 2. Distribution of PTHrP concentrations in premenopausal (n=15) and postmenopausal (n=45) women.

10 3.5 A 2 B PTHrP, pmol/l R² = PTHrP, pmol/l R² = BMI BMI Supplemental Figure 3. Association of PTHrP concentration with BMI in women (A, n=60) and men (B, n=53)