Effects of pretreatment protocols on human amniotic fluid protein profiling with SELDI-TOF MS using protein chips and magnetic beads

There is increasing interest in the use of human amniotic fluid (AF) proteomics with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) for diagnosing pregnancy-associated abnormalities. A critical parameter of diagnostic biomarkers is the accuracy and reproducibility of protein patterns. We evaluated the effects of common pretreatment protocols on protein patterns generated using SELDI mass spectrometry with two different protein capture strategies (including functional protein chips and functionalized magnetic beads prior to MS analysis) in AF. Various extrinsic factors involved in processing and storing amniotic fluid, including matrix composition, sample storage time, temperature and freeze–thaw cycles, were analyzed regarding their impact on AF protein patterns using SELDI mass spectrometry with 2 different protein capture strategies. Three extrinsic factors (sample storage for 3 days at either room temperature or 4 °C or freeze–thawing the sample 5 times) significantly decreased the number or intensities of protein peaks detected in AF. Matrix dilutions also changed the spectra of AF, with more peaks and higher intensities observed with 50% α-cyano-4-hydroxycinnamic acid (CHCA). Moreover, protein chips captured more proteins or peptides than magnetic beads on SELDI-TOF MS profiling of AF. These results suggest that extrinsic factors must be taken into account for valid data interpretation to ensure good reproducibility of AF profiling by SELDI mass spectrometry.