Proteome profiling of cell-free coelomic fluid from the common starfish Asterias rubens
Echinoderms, possessing outstanding regenerative capability, provide unique model system for the study of the response to injury. However, there is little known about the proteomic composition of coelomic fluid, an important biofluid circulating through the whole body and reflecting an overall biological status of the organism. In this study, we used LC-MALDI tandem mass spectrometry to characterize proteome of cell-free coelomic fluid of starfish Asterias rubens and follow the changes occurring in response to puncture wound and blood loss. Our study demonstrated significant changes of CF proteome during the first hours after injury and presented a series of candidate proteins involved in early response to injury, providing interesting targets for future functional studies.
Sample Processing Protocol
The samples of CF were collected 6 hours after puncture wound (PW6h), and 6 and 72 hours after blood loss, BL6h and BL72h, respectively. Samples collected just before injury served as control (CPW and CBL). The CF was obtained from coelomic cavity by puncturing aboral epidermis at the arm tip with 21G two-sided needle and collecting 1,5 ml of the CF by gravity flow into microtube containing 45 μl of 0.5 M EDTA, pH 7.5. Two low-speed centrifugations were performed to gently separate CF from circulating cells, followed by filtration with Spin-X centrifuge tube filters (0.45 μm). The flow-through aliquots of 500 μl from independent samples of each experimental group were pooled (n = 12 for PW, n = 8 for BL) giving rise 6 ml and 4 ml of cell-free CF respectively. Pooled CF samples were subjected to solid-phase extraction using Oasis HLB CC (Waters) and Strata C18-T 100 mg (Phenomenex) solid-phase extraction tubes sequentially connected. In-solution digestion was performed according to DOC protocol with trypsin/Lys-C mix (Promega). All digests were then desalted and concentrated with solid-phase extraction tips packed with 20 mg of Strata C18-T sorbent (Phenomenex). Each sample was rehydrated, filtered and divided into four 50 μl aliquots, used as technical replicates in LC-MALDI-MS/MS analyses. Peptides were separated with a Jupiter Proteo C12 reversed-phase column (1 mm × 50 mm, 4 μm, 90 Å, Phenomenex) on a microbore HPLC system (MiLiChrom A-02, EcoNova). A sample volume of 50 μl was injected and separated using linear gradient of 10-35% B over 54 min followed by 35-90% B for 6 min at a flow rate of 50 μl•min-1. The mobile phases used were A, 0.125% (v/v) TFA in water and B, 0.125% (v/v) TFA in acetonitrile. Column was thermostated at 45 °C. Micro-fraction collector was used to deposit a total of 912 fractions of 0.5 μl in a 24 × 38 array on a LC-MALDI plate (SCIEX). The fractionated samples were analysed with a (TOF/TOF 5800 System, SCIEX) instrument operated in the positive ion mode. The MALDI stage was set to continuous motion mode. MS data was acquired at 2400 laser intensity with 1000 laser shots/spectrum (200 laser shots/sub-spectrum) MS/MS data was acquired at 3300 laser intensity with DynamicExit algorithm and high spectral quality threshold or a maximum of 1000 laser shots/spectrum (250 laser shots/sub-spectrum) Up to 35 top precursors with S/N > 30 in the mass range 850 – 4000 Da were selected from each spot for MS/MS analysis.
Data Processing Protocol
Repeated measure design was used to reduce biological variance among individuals, therefore control and time-point samples were taken from the same individual. Two traumatic treatment (PW and BL) and five experimental groups (i.e. CPW, PW6h, CBL, BL6h and BL72h) each with one pooled biological replicate (12 individuals in PW, and 8 individuals in BL) were analyzed yielding a list of identified CF proteins. Four independent LC-MALDI TOF/TOF acquisitions of each pooled sample were performed and processed together in one run with the Protein Pilot 4.0 software (SCIEX). The Paragon algorithm 4.0 was used in thorough mode with biological modifications and substitutions enabled. Carbamidomethyl cysteine was set as fixed modification. The subject of Paragon searches was a pooled protein database comprising protein-coding open reading frames (ORFs) predicted from three A. rubens transcriptome shotgun assembly datasests: ovary (Reich, Dunn et al. 2015), tube foot (Hennebert, Leroy et al. 2015), and radial nerve (Semmens, Mirabeau et al. 2016). Each transcriptome shotgun assembly dataset was subjected to two iterative reassemblies with iAssembler. Then, ORFs were predicted with TransDecoder-v5.0.2 at minimum ORF length of 70 amino acids and using homology option. Searches with HMMSCAN against Pfam-A version 31.0, and with BLASTP against set of Echinodermata sequences downloaded from UniProtKB on December 12th 2017, was used for homology option. Redundant sequences were then removed using CD-HIT at 97% identity threshold, local alignment and 80% alignment coverage for shorter sequence (-c 0.97, -G 0 and -aS 0.8 options), producing 24,796 ORFs. These were then enriched with a set of 947 non-redundant secretory and single-pass transmembrane protein-coding sequences predicted from ovary transcriptome shotgun assembly of Asterias forbesi as described above. Resulting set filtered with CD-HIT produced final database of 25,725 protein-coding sequences. The database also incorporated a list of common contaminants. False discovery rate (FDR) analysis was done by analysis of reversed sequences using the embedded PSEP tool.
Sergey Shabelnikov, Institute if Cytology of RAS
Sergey Shabelnikov, Department of intracellular signalling and transport, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia ( lab head )