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The host cell proteome of Physcomitrella patens
Host cell proteins are inevitable contaminants of biopharmaceuticals. Here, we performed detailed analyses of the host cell proteome of moss (Physcomitrella patens) bioreactor supernatants using mass spectrometry and subsequent bioinformatics analysis. Distinguishing between the apparent secretome and intracellular contaminants, a complex extracellular proteolytic network including subtilisin-like proteases, metallo-proteases and aspartic proteases was identified. Further, we confirmed predicted cleavage sites of 40 endogenous signal peptides employing an N-terminomics approach.
Sample Processing Protocol
Sample preparation: Supernatants of one 100 litre reactor (Wave_100) and two samples from 10 litre reactors (Wave_10A, Wave_10B) were provided as ultraconcentrate. The supernatant of the 100 litre reactor was concentrated to 40 ml and buffer was exchanged to 100 mM Tris, 2.5M NaCl, pH 7.5 using a Cross-Flow (Centramate 500s, Pall, Crailsheim, Germany) with a 10 kDa cut off (Centramate Delta cassette, Pall). The ultrafiltrate was divided into aliquots of 5 ml and each was precipitated with acetone. The supernatants from the two 10 litre reactors were concentrated each to 170 ml and aliquots of 35 ml were precipitated overnight using 10% trichloroacetic acid (TCA). The aliquots were centrifuged at 20,000 x g for 15 min at 4°C. The supernatant was discarded, and the remaining pellet was washed with acetone containing 0.2% DTT. After centrifugation an additional washing step using acetone without DTT was performed. The remaining protein pellets were air dried and stored at -20°C for further use. Protein pellets were dissolved in 100 mM HEPES-NaOH, pH 7.5, 0.2% SDS and the protein concentration was measured using the BCA assay. 50 µg dissolved proteins were mixed with an equal volume of Laemmli buffer (Bio Rad, Munich, Germany). Reduction of cysteine residues was carried out using Reducing Agent (Life Technologies™, Carlsbad, USA) 1:10 at 95°C for 10 min. Alkylation was performed at a final concentration of 100 mM iodacetamide for 20 min at RT. SDS-PAGE was performed with 12% or 7.5% Mini-Protean® TGXTM gels (Bio Rad). After electrophoresis, gels were stained with PageBlue (Thermo Scientific, Waltham, USA) as recommended by the manufacturer. Each lane was cut into 15 gel slices. Preparation of the slices and trypsin digestion was done as described (Hoernstein et al., 2016, doi: 10.1074/mcp.M115.057190). Enrichment of N-terminal peptides: Enrichment of N-terminal peptides was performed using HCP precipitate from the 100 liter wave reactor. Here we employed three different proteases (trypsin, GluC, chymotrypsin). The dimethylation was performed as described (Hoernstein et al. 2016, doi: 10.1074/mcp.M115.057190). Protein pellets were dissolved in binding buffer (McDonald and Beynon 2006, DOI:10.1038/nprot.2006.317) containing 20 mM NaH2PO4, 150 mM NaCl pH 7.5 with 0.2% SDS and in-solution digest using either trypsin (Promega, Madison, USA), GluC (Thermo Scientific) or chymotrypsin (Promega) was performed at an enzyme-to-substrate ratio of 1:25 for 4 h at 37°C (trypsin, GluC) or 25°C (chymotrypsin). Then the ratio was increased to 1:20 and the reaction was carried out overnight. Enrichment of amino-terminal labelled peptides was carried out using 200 µl NHS-sepharose slurry (GE Healthcare). The slurry was centrifuged for 30 sec at 200 x g. The supernatant was discarded and 400 µl ice-cold 1 mM HCl was added. The slurry was centrifuged again and the supernatant was discarded. Afterwards the sepharose was washed with 1 ml binding buffer without SDS. The samples were applied to the prepared sepharose and incubated for 4h at RT. The sepharose was again centrifuged and the supernatant was transferred to a new tube containing freshly prepared sepharose. The used sepharose was washed with 20 μl binding buffer and the supernatant was also added to the freshly prepared sepharose. The enrichment reaction was carried out overnight at 4-8°C. The enriched peptides were desalted using 200 µl C18 StageTips (Thermo Scientific) that were supplemented with an additional layer of EmporeTM SPE Disk C18 material (Sigma Aldrich). The tips were washed prior to use with 100 µl 0.1% trifluoroacetic acid (TFA) and subsequently with 100 µl 80% acetonitrile (ACN), 0.1% TFA. The tips were again equilibrated with 100 µl 0.1% TFA and the samples were loaded afterwards. The remaining sepharose was washed with 50 µl binding buffer and the supernatant was also transferred to the tip. The tips were washed with 100 µl binding buffer and the retained peptides were eluted with 100 µl 80% ACN, 0.1% TFA. The eluate was vacuum dried and the dried peptides were directly used for MS/MS analysis.
Data Processing Protocol
Raw data processing and database search: Raw data processing was performed using Mascot Distiller V18.104.22.168 (Matrix Science, Boston, Massachusetts, USA). Database searches on the processed raw data were performed using Mascot Daemon V2.4 (Matrix Science) against the Physcomitrella patens database containing all version 1.6 protein models (Zimmer et al. 2013, doi: 10.1186/1471-2164-14-498) as well as their reversed sequences used as decoys and simultaneously against an in-house database containing all sequences of known typical contaminants (e. g. human Keratins, Trypsin, 267 total entries, available on request). For database searches of dimethylated samples the fixed modifications were carbamidomethyl (C) +57.021464 Da and 13C,d2 dimethyl (K) +34.063117 Da. Variable modifications used for database search were Gln >pyro Glu (N term Q) -17.026549 Da, oxidation (M) +15.994915 Da, acetyl (N-term) +42.010565 Da, 13C,d2 dimethyl (N-term) +34.063117 Da, hybrid-methylation (N-term) +31.047208 Da, formyl (N-term) +27.994915 Da. For database searches of the in-gel digested samples the fixed modifications were carbamidomethyl (C) +57.021464 Da. Variable modifications used for database search were Gln >pyro Glu (N term Q) -17.026549 Da, oxidation (M) +15.994915 Da, acetyl (N-term) +42.010565 Da, hydroxyproline (P) +15.994915 Da, and phospho (ST) +79.966331 Da. For all searches the peptide mass tolerance was ± 8 ppm and the fragment mass tolerance was set to ± 0.02 Da. For database searches on trypsin-digested samples the enzyme specificity was set to semitryptic with a total of 2 in the case of in-gel digested samples or 5 missed cleavage sites in the case of in-solution digested samples. For samples digested with GluC or chymotrypsin the specificity was set to “none”. All Mascot searches were loaded into Scaffold 4 (Version 4.2.1, Proteome Software, Portland, Oregon, USA). Database searches from amino-terminal labelled samples were additionally searched using X!Tandem implemented in Scaffold 4 against the P. patens protein models V1.6 with the same search parameters as used for the Mascot searches. Samples were loaded and analysed using the legacy PeptideProphet scoring (high mass accuracy) with standard experiment wide protein grouping. Results were either filtered using the Protein- and PeptideProphetTM 29,30 or directly with the FDR filter implemented in Scaffold 4 software. The filter settings for the SDS-PAGE samples were: Protein threshold: 99% ProteinProphetTM, peptide threshold 95% PeptideProphetTM, minimum number of peptides: 2 and for the measurements of enriched N-terminal peptides: Protein threshold: 1% FDR, peptide threshold: 0.5 FDR, minimum numbers of peptides: 1.
Ralf Reski, Faculty of Biology, University of Freiburg (Chair Plant Biotechnology), Schaenzlestr. 1, D-79104 Freiburg
Ralf Reski, Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestr.1, D-79104 Freiburg, Germany ( lab head )
Hoernstein SNW, Fode B, Wiedemann G, Lang D, Niederkrueger H, Berg B, Schaaf A, Frischmuth T, Schlosser A, Decker EL, Reski R. The host cell proteome of Physcomitrella patens harbours proteases and protease inhibitors under bioproduction conditions. J Proteome Res. 2018 PubMed: 30226384
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