MALDI-TOF/MS PROTEOMIC SIGNATURE OF PRE-ANALYTICAL SAMPLE QUALITY

Background: Pre-analytical issues represent a critical aspect for proteomic biomarker discovery studies. Pre-analytical processing (sample types, centrifugations, temperatures and storage duration) was studied to identify MALDI-TOF/MS signatures of sample quality. Materials and methods: 400 mL blood was collected in serum, EDTA and citrate tubes from one donor. Tubes were kept at room temperature (RT) or refrigerated (CT). After 30 minutes (baseline), 3 hours (hrs), 6 hrs and 9 hrs, samples were centrifuged once (1200 g, 10 minutes) ortwice (2500 g, 15 minutes). 100 uL of each sample were precipitated by ACN (1:1v/v). Peptides were dried, resuspended in 0.1%TFA, ZipTip desalted and analyzed at MALDI-TOF/MS. Spectra were processed by mMass; relative intensities (RI) were used to normalize signals. For each anticoagulant and storage condition, the most abundant features were evaluated at each time-point. For the most variable features, their baseline RIs were compared to those obtained after 9hrs of storage. Results: Features in citrate tubes did not significantly varied. At RT, EDTA tubes centrifuged once, the feature at m/z 1896 showed a RI increase over storage time (from 80% to 120%). In serum tubes, RT storage showed a decreasing trend of the m/z 1206 and 1350 features. By comparing the most variable features at baseline with respect to 9 hrs storage, no significant differences were found in EDTA tubes; in serum tubes, the feature at m/z 2021 dramatically increased after 9 hrs of storage at RT (50 and 200 fold-increase, for 1 or 2 centrifugations, respectively). A lower increase was observed at CT conditions (10 and 20 fold-increase, for 1 or 2 centrifugation, respectively). Conclusions: EDTA and citrate plasma seem to be less influenced by storage conditions. MALDI-TOF/MS features at m/z 1206, 1350 and 2021 could be indicators of serum specimen quality, especially when RT storage is adopted.