Comparative proteome stability analysis of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human colon mucosal biopsies
Clinical proteomics research is in part limited by the availability of clinical samples. However, large biobanks exist worldwide containing formalin-fixed and paraffin embedded samples and samples stored in RNAlater. The extraction of proteins for proteome analysis from samples prepared by either method has been demonstrated. However, the impact of the preservation method on the result of a quantitative proteome analysis remains largely uninvestigated. We, therefore, conducted a proteome analysis of human colon mucosal biopsies where the material had been preserved accordingly. Twenty-four colon mucosal biopsies were extracted from the sigmoideum by endoscopy from two participants. The biopsies were either directly-frozen, stabilized in RNAlater, or stabilized by formaldehyde fixation immediately or incubated for 30 min to simulate a clinical situation, followed by paraffin embedding. The impact of the preservation method on the result of a proteome analysis was characterized by high throughput gel free quantitative proteomics.
Sample Processing Protocol
Collection of Sample Material The project was approved by The Regional Scientific Ethical Committee (S-20120204) and the Danish Data Protection Agency (2008-58-035), and all participants had given informed consent to participate in the study. Colon mucosal biopsies were sampled from the sigmoideum of two healthy persons without findings, by endoscopy at Aabenraa Regional Hospital Denmark. Twelve biopsies were extracted from each person approximately 40 cm from the anus. Directly frozen (DF) biopsies were immediately transferred to individual cryotubes and within 10-20 seconds snap-frozen with liquid nitrogen followed by storage at -80°C for one month. Similarly, biopsies were immediately transferred to individual cryotubes prefilled with 500 µL RNAlater (Life Technologies, Carlsbad, CA, USA), incubated at room temperature for 24 h followed by storage at -80°C for one month. FFPE biopsies were within seconds following extraction placed in preservation cartridges and immediately (iFFPE), or after 30 minutes at ambient temperature (sFFPE), stabilized in 4% formaldehyde and stored at room temperature for one week prior to paraffin embedding. FFPE sample preparations were performed at Department of Pathology at Aalborg University Hospital, Denmark according to current standards. Proteomics – Sample Preparation DF and RNAlater preserved materials were processed directly, by addition of 500 µL lysis buffer (12 mM sodium deoxycholate (SDC), 12 mM SDS in 300 mM Tris/HCl, pH 9.0). FFPE tissues and FFPE bone material were extracted using a scalpel. The FFPE samples were deparaffinized and rehydrated by washing in xylene (3x), and in 100% ethanol (2x), 96% ethanol (2x), 70% ethanol (2x), water, and 500 µL lysis buffer was added. All samples were homogenized using a Bullet Blender Gold (Next Advance Inc., Averill Park, NY, USA). The samples were incubated at 95°C, 60 min for FFPE samples and 10 min for DF and RNAlater samples, and sonicated for 10 min. A modified FASP protein digestion with trypsin was performed, with phase inversion surfactant removal. For each sample, a volume corresponding to 100 µg protein was transferred to individual YM-30 kDa spinfilters (Millipore, Billerica, MA, USA) and centrifuged. All centrifugation steps were performed at 14,000 g for 15 min at 4°C. Protein disulfide bonds were alkylated with 12 mM tris(2-carboxyethyl)phosphine (Thermo Scientific, Waltham, MA, USA) for 30 min at 37°C, and reduced with 50 mM chloroacetamide (Sigma-Aldrich, St. Louis, MO, USA) for 20 min at 37°C in the dark. The reducing and alkylating agents were dissolved in 120 mM SDC in 50 mM triethylammonium bicarbonate (TEAB), pH 8.5, and centrifuged after each step. In preparation for digestion, 400 µL digestion buffer (12 mM SDC in 50 mM TEAB) was added to the spinfilter and centrifuged. A 1:50 (w/w) trypsin:protein ratio dissolved in 50 µL digestion buffer was added to the spinfilter, and the samples were digested overnight at 37°C. The flow-through containing the peptides was retrieved by addition of 50 µL digestion buffer and centrifugation. To facilitate SDC removal, a phase separation performed with 3:1 (v/v) ethyl acetate:sample, acidified by addition of formic acid (FA) to a final concentration of 0.5%. Total phase separation was achieved by 2 min agitation followed by centrifugation. The aqueous phase was collected and vacuum centrifuged overnight and stored at -80°C until time of analysis. Five µg total peptide material was analyzed per LC-MS analysis, in a random sample order. The samples were analyzed using a UPLC-nanoESI MS/MS setup with an UltiMate 3000 UHPLC system (Dionex, Sunnyvale, CA, USA) upgraded with a RSLC nanopump module. The system was coupled online with an emitter for nanospray ionization (New objective picotip 360-20-10) to a Q Exactive Plus mass spectrometer (Thermo Scientific, Waltham, USA). The peptide material was loaded onto a 2 cm trapping reversed phase Acclaim PepMap RSLC C18 column (Dionex) and separated using an analytical 50 cm column (Dionex) kept at 40°C. The sample was eluted with a gradient of 96% solvent A (0.1% FA) and 4% solvent B (0.1% FA in ACN), which was increased to 10% solvent B on a 1 minutes ramp gradient at a constant flow rate of 300 nL/min. Subsequently, the gradient was raised to 30% solvent B on a 180 min ramp gradient. The mass spectrometer was operated in positive mode, selecting up to 12 precursor ions with a mass window of m/z 1.6 based on highest intensity for HCD fragmenting, at a normalized collision energy of 27. Selected precursors were dynamically excluded for fragmentation for 30 sec.
Data Processing Protocol
A label-free quantitation analysis of the biopsy LC-MS rawfiles was performed in MaxQuant 126.96.36.199 by searching the data-files against the Uniprot Homo sapiens reference proteome (UP000005640, last modified 2015-01-16, protein count 68,015) attached to this submission. All standard settings were employed with carbamidomethyl (C) as a static modification and protein N-terminal acetylation, deamidation (NQ), oxidation (M) and peptide N-terminal formylation were included as variable modifications. All proteins are reported <1% FDR, to ensure high-confidence protein identifications.
Bennike TB, Kastaniegaard K, Padurariu S, Gaihede M, Birkelund S, Andersen V, Stensballe A. Proteome stability analysis of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human colon mucosal biopsies. Data Brief. 2016 Feb 6;6:942-7. eCollection 2016 Mar PubMed: 26937473