Serum samples
A QC sample was prepared by pooling serum from 10 non-fasting anonymous donors (CDC Institutional Review Board approval 1652), dispensed into single use aliquots and stored at -80°C. This QC sample was fractionated using the Biomek 2000 Automation Workstation (Beckman Coulter) and the Expression Difference Mapping Kit-Serum Fractionation according to the manufacturer (Ciphergen). Six fractions were collected and stored at -80°C for 2 to 11 days keeping storage time the same for the equivalent fractions of each sample. The fractionation procedure was repeated on the same QC sample three times with 2 weeks in between each fractionation. Each fractionation procedure representing one batch (Fig. 1).
ProteinChips
To load the different ProteinChips, we used the correspondent Biomek 2000 Automation Workstation methods. The IMAC 30 ProteinChip was loaded with copper sulfate (Ciphergen Expression Difference Mapping kits IMAC buffer), and H50 ProteinChip (Ciphergen Expression Difference Mapping kit-H50 buffer) was washed with 50% acetonitrile before being loaded with the serum fraction. Except for these modifications, treatment of the ProteinChips included two pre-washes of 150 μl of corresponding buffer for 5 minutes each followed by addition and 1 hour incubation on a microplate shaker of 100 μl sample (10 μl serum fraction in 90 μl of buffer). Each protein chip had three 150 μl stringency washes followed by 2 quick washes with 200 μl of HPLC water. One binding-washing buffer was used for IMAC-Cu and H50 ProteinChips, and a low stringency (LS) and high stringency (HS) binding-washing buffer was used for the CM10 ProteinChip (resulting in 4 spectra for each serum fraction). After the last wash, the ProteinChips were air-dried for 20 minutes. The energy absorbing molecule (EAM) was sinapinic acid (5 mg in 200 μl acetonitrile, 200 μl of 1% trifluoroacetic acid), which was freshly prepared before a double application (1 ul each) to all ProteinChips with exactly 15 minutes drying time between each application [6]. In Exp 1 the EAM was applied by hand with a one-channel pipette. In the optimized method (Exp 2) it was applied using the Biomek 2000 robot, which dispensed 1 μL of EAM simultaneously to the eight spots of a chip. The differences between the 2 experiment protocols are outlined in Table 3. Mass analysis was performed using a PBSIIc mass spectrometer over an m/z range of 3000–30000.
Acquisition protocols
For both studies, we optimized the spot protocols using the QC sample for the mass range between 3000 and 30000 Da. The major difference between Exp 1 and Exp 2 are the acquisition protocols. For Exp 1, spot protocols were optimized for whole serum on each different ProteinChip. To establish the optimized protocol different laser intensities and detector sensitivities were used for the collection of the spectra, and visual inspection used to assess the best spectrum. These parameters were then used in the spot protocol for experimental data acquisition. In Exp 2, spot protocols were optimized for each serum fraction-ProteinChip combination by adjusting laser intensity and detector sensitivity. The spectra collected and processed using Ciphergen Express™ software (version 3) (CE). All spectra normalized by total ion current and calibrated. Peak detection performed using with peak height of 10 and valley depth of 5. Spectral quality was assessed using 2–3 randomly selected peaks by comparing peak intensity, S/N and resolution. As Semmes et al. described [8], the laser intensity and the detector sensitivity for the spot protocol were chosen to increase the peak detection and resolution without increasing the signal to noise ratio. The optimized laser and detector sensitivity settings were used in the appropriate spot protocols. We did not change the detector voltage during the course of a study; but we changed it between the 2 experiments after optimization by DL Vary performance check as recommended by Ciphergen Biosystems. In Exp 1 the mass spectra were derived from 10 shots per transient, with a spacing of 5 between transients, acquiring a total of 130 laser shots for each spectrum. This was after 2 warming shots not included in the spectrum. In Exp 2, a total of 192 shots were collected for each spectrum, from 12 transients every 4 positions after 2 warming shots not included in the spectrum file.
Instrument performance evaluation
QC and performance checks included calibration and alignment of the Biomek 2000 performed monthly. Mass accuracy, resolution and sensitivity of the spectrometer were evaluated monthly using the insulin standard chip and the bovine IgG standard chip (Ciphergen). A normal phase ProteinChip, NP20 (Ciphergen) was run weekly, loaded with All-in-1 Protein standard II for external calibration of the spectra. To minimize slight systematic shifts in the time-of-flight data from one spot to another one, we used the CE to calculate a spot-to-spot correction factor. The correction factor was calculated from 8 spectra (One spectrum per spot position) of All-in-I Peptide Standard (Ciphergen) on NP20 ProteinChip.
Data Analysis
All data were analyzed in CE. We applied baseline smoothing before fitting the baseline using a moving average filter window of 25 points, and an automatic fitting width. We used an average filter of 0.2 times expected peak width, to remove high frequency noise from the spectrum improving the S/N. Spectral intensities were normalized by total ion current and spectra with normalization factor > 2SD were excluded. The spectra were calibrated from a weighted 3 parameter quadratic equation calculated from 4 protein standards (mass range 7 to 30 kDa). Prior to alignment we did peak detection with settings of peak height and valley depth at 6 times the noise. Peak alignment was performed using the following settings: 0.2% of mass window and minimum S/N of 5. Peaks were identified using the CE Biomarker Analysis Module Cluster Wizard according to these settings: first pass S/N ≥ 3 and valley depth ≥ 3, minimum peak threshold 80% of all spectra, preserving all 1st pass peaks, mass window 0.2% of mass, second pass S/N ≥ 2 and valley depth ≥ 2, add estimated peaks to complete clusters, autocentroid, and m/z range 3000–30000.
Calculations of average CVs for peak intensity were accomplished in Microsoft Excel. Statistical analyses using a non-parametric Kruskal-Wallis test, with a bootstrap of 2000 randomizations for multiple test correction, were performed using Partek Genomics Suite (version 6.2 Copyright © 2006).
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