Introduction Proteomic quantification methods have developed significantly, due to the use of isotope labelling. More recently label-free methods have been developed to allow faster and simpler analysis. We have conducted a proof-of-principle experiment to determine the applicability of this method to pregnancy plasma. We have then applied this technique to early pregnancy samples from women with and without preterm delivery.
Methods Five replicate plasma samples containing myoglobin spiked at different concentrations were depleted of abundant proteins using immunodepletion (MARS-14, Agilent) and fractionated into six fractions using reverse-phase chromatography (Agilent). Each fraction was digested and run on an LC-QTOF (Agilent) platform to acquire label-free MS data. Data were analysed using Progenesis (Non-linear Dynamics). Replicate samples were used to assess technical variability and to determine the ability of the software to identify quantitative differences in myoglobin levels. Samples taken at 20 weeks from 12 women with preterm labour and 12 controls from the SCOPE study were analysed in a randomised, blinded order.
Results Myoglobin spiked into replicate plasma samples at either 1 or 2µg/ml (n=5, duplicate LC-MS injections) was identified as the only differentially abundant protein (p<0.001, ANOVA). Importantly, no false positives were identified as no other proteins reached p<0.05 significance. Analysis of the clinical samples revealed >500 protein identifications; quantitative analysis is ongoing.
Conclusion Protein level chromatography coupled with label-free quantification using mass spectrometry is complementary to isotope-labelled methods in the identification of differentially abundant proteins in plasma samples. This technique has the potential to identify new markers for preterm labour.
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