Most proteomics studies using mass spectrometry, which can analyze multiple proteins (proteome) simultaneously, examine one blood specimen per participant; however it is unknown how well measures at one point in time reflect an individual’s long-term proteome pattern. Therefore, we examined the reproducibility of the proteome in postmenopausal women over 3-years, using surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF). In 1989-1990, 32,826 women from the Nurses’ Health Study provided a blood specimen. A subset of women provided additional blood samples yearly for two years after the initial blood draw. We randomly selected 60 women who provided a blood sample in years 1 and 3. Women drew blood into heparin plasma tubes and shipped the sample, with an icepack, by overnight courier; samples were processed within 6 hrs of receipt. Samples were collected in the early morning after fasting for >10 hrs. Previous pilot studies suggested that the blood collection protocol did not substantially affect the results of the SELDI-TOF assay. This assay detects protein peaks based on a spectrum generated when plasma is spotted on a chip surface and subjected to energy transfer (low or high protocol) by a nitrogen laser beam. Four conditions were examined: unfractionated plasma on a CM10 and H50 chip, pH≥9 plasma fraction on a CM10 chip, and the organic fraction on the H50 chip. Initial peak detection occurred in a reference sample containing a mixture of all plasma samples, using a varying signal-to-noise ratio (SNR; 2.0 or 3.0). Participant and quality control (QC) samples were aligned to the reference sample and peak intensity in these samples was assessed for all peaks identified in the reference sample. We calculated coefficients of variation (CV) using 43 blinded QC samples from 13 donors. Mixed models were used to determine the intraclass correlation (ICC) of peak intensities over 3 years. The average CVs within conditions ranged from 16% (H50, organic fraction, low protocol) to 64% (CM10, PH≥9 fraction, high protocol). In general there was a strong correlation between the CV and mean peak intensity of the QC samples (median=-0.48), such that values <0.2-2.5 had poor reliability. The mean ICC within conditions ranged from 0.40 (H50, unfractionated, low protocol) to 0.68 (CM10, unfractionated, high protocol). For a SNR cutoff of 2.0, we assessed the ICC for 333 peaks, of which 248 (74%) had an ICC≥0.40. For a SNR cutoff of 3.0, we assessed the ICC for 188 peaks, of which 118 (63%) had an ICC≥0.40. In conclusion, our results suggest that protein peak reproducibility over three years was reasonable among postmenopausal women, such that one sample may reflect the proteome pattern over time. However, future studies should consider eliminating peaks with poor CVs or a low ICC by including multiple QC and reproducibility samples.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA