| Metadata |
| datasetIdentifier | PASS00557 |
| datasetType | MSMS |
| submitter | Qiyao Li <qli@chem.wisc.edu> |
| submitter_organization | Univ. of Wisconsin-Madison |
| lab_head_full_name | Lloyd M Smith |
| lab_head_email | smith@chem.wisc.edu |
| lab_head_organization | Univ. of Wisconsin-Madison |
| lab_head_country | United States |
| datasetTag | CollMatriDecellliver |
| datasetTitle | Rat tail collagen I gel; Growth factor reduced Matrigel; Decellularized rat liver |
| publicReleaseDate | 2015-09-28 00:00:00 |
| finalizedDate | |
| summary | Proteomic analyis of rat tail type I collagen gel, growth factor reduced (GFR) Matrigel, and rat livers after decellularization by vascular perfusion. |
| contributors | Qiyao Li, Sinan Ozer, Brian L. Frey, Basak E. Uygun, Nathan V. Welham, Lloyd M. Smith |
| publication | unpublished |
| growth | |
| treatment | 1. Thaw harvested rat livers overnight at 4oC.
2. Fill the perfusion system with PBS and keep it running for 10 minutes. Fill PBS into a 10-cm petri dish.
3. Reduce the flow rate of PBS to 1 ml/min.
4. Carefully transfer the thawed liver to the PBS filled 10-cm petri dish.
5. Make sure there is no air inside the catheters. Fill with PBS using a syringe and a needle to remove any bubbles.
6. Carefully connect the PBS flowing tube to the catheter.
7. Record date and time. Continue with PBS perfusion overnight.
After overnight PBS wash; t = 18 h (d1)
8. Start perfusion with 0.01% SDS. Keep it for 5 mins.
9. Perfuse with 1X PBS for 1 hour.
10. Perfuse with 0.01% SDS for 10 mins.
11. Perfuse with 1X PBS for 1 hour.
12. Perfuse with 0.01% SDS for 15 mins.
13. Perfuse with 1X PBS for 1 hour.
14. Perfuse with 0.01% SDS for 20 mins.
15. Perfuse with 1X PBS for 1 hour.
16. Perfuse with 0.01% SDS overnight.
#2 Half decell liver; After wash with 0.01% SDS; t = 40 h (d2)
17. Perfuse with 0.1% SDS for 24 hours.
After wash with 0.1% SDS (d3-1)
18. Perfuse with 0.2% SDS for 3 hours. Go to step 20 if the effluent is clear and the liver is transparent.
19. Perfuse with 0.5% SDS for 3 hours. Go to step 20 if the effluent is clear and the liver is transparent.
20. Perfuse with dH2O for 15 minutes.
21. Perfuse with 1% Triton X-100 for 30 minutes.
22. Perfuse with 1X PBS for 1-2 hours as a final wash. |
| extraction | For rat tail type I collagen or GFR Matrigel (both from BD Bioscience, San Jose, CA), aliquots of approximately 200 µg protein was added to 150 µL of SDT solution containing 4% SDS, 0.1 M Tris-HCl (pH 7.6) and 0.1 M dithreothreitol (DTT) (all reagents from Sigma-Aldrich, St. Louis, MO). For decellularized or intact tissues, aliquots of 15 mg tissue were washed with ice cold PBS solution (Invitrogen, Grand Island, NY) and ground with disposable pellet pestles (Kimble Chase Kontes, Vineland, NJ) for 1 min in 1.5-ml tubes and 150 µL SDT solution was added. Sample was then heated at 95 °C for 7 min. and sonicated on ice with a probe sonicator (Misonix XL2015, Misonix microtip PN/418, Farmingdale, NY)—alternating 20 seconds on and 20 seconds off for 6 min, followed by centrifugation at 22 °C for 5 min. at 16,100 g. |
| separation | For GFR Matrigel, off-line high-pH RPLC peptide fractionation was performed, on a HPLC system (Shimadzu, Columbia, MD) using a C18 Gemini 3μ, 110Å, 3.0x150mm column (Phenomenex, Torrance, CA). Mobile phase A was 20 mM ammonium formate and mobile phase B was 20 mM ammonium formate in 70% ACN. The HPLC flow rate was 1 mL/min and the gradient last for 20 min with increasing mobile phase B. The eluent was pulled into 6 fractions, evaporated to dryness in the SpeedVac and reconstituted in 5% ACN, 2% FA. |
| digestion | The FASP protocol was used for SDS removal and on-filter digestion. Briefly, a 30 μL aliquot of the supernatant was added to a 30K MW Vivacon 500 filter (Sartorius, Bohemia, NY), washed, alkylated, and digested with trypsin (Promega, Madison, WI; protein:enzyme ratio of 50:1) overnight at 37 °C. Finally, the digest was collected by centrifugation. |
| acquisition | Approximately 0.6 µg protein digest, estimated by BCA protein assay, was injected into a Waters nanoAcquity HPLC coupled to an ESI ion-trap/orbitrap mass spectrometer (LTQ Orbitrap Velos, Thermo Scientific, Waltham, MA). Peptides were separated on a 100 μm inner diameter column packed with 20 cm of 3 μm MAGIC aqC18 beads (Bruker-Michrom, Auburn, CA), and eluted at 0.3 µL/min in 0.1% FA with a gradient of increasing ACN over 2.5 h. For running the rat liver samples, the column was packed with 20 cm of 1.7 μm BEH C18 particles (Waters, Milford, MA), and a heater cartridge was used to keep the capillary column at 60 °C. A full-mass scan (300-1500 m/z) was performed in the orbitrap at a resolution of 60,000. The ten most intense peaks were selected for fragmentation by high-energy collisional dissociation (HCD) at 42% collision energy, with a resolution of 7500, and isolation width of 2.5 m/z. Dynamic exclusion was enabled with a repeat count of 1 over 30 s and an exclusion duration of 120 s. |
| informatics | The acquired raw files were analyzed by MaxQuant (30, 31) (version 1.4.1.2). The derived peak lists were searched with Andromeda (32) against the appropriate organism Uniprot canonical protein databases (Rattus norvegicus: 7853 reviewed sequences downloaded on May 4, 2013 for rat tail collagen search, and 33,607 sequences (reviewed plus unreviewed) downloaded on May 29, 2014 for rat liver searches; Mus musculus: 16,642 reviewed sequences downloaded on December 16, 2013; Homo sapiens: 20,278 reviewed sequences downloaded on December 5, 2013) supplemented with 262 common contaminants. Precursor masses and fragment ions mass tolerances were set to 4.5 ppm and 20 ppm respectively. Static cysteine carbamidomethylation (+57.0215 Da) and up to 5 variable methionine and proline oxidation (+15.9949 Da) were specified. The modification parameters were a little different for the collagen gel search: oxidation of lysine (+15.9949 Da) and glycosylation of lysine (monosaccharide, +178.0473 Da, disaccharide, +340.0995 Da) were also included as variable modifications and up to 7 variable modifications were allowed. A false discovery rate of 1% at both the peptide and the protein level was allowed. Up to two missed cleavages were allowed and a minimum of two unique peptides per protein was required. Protein groups containing matches to proteins from the reversed database or contaminants were discarded. Intensity-based absolute quantification (iBAQ) as well as label-free quantification (LFQ) algorithms embedded in the MaxQuant software package were selected for quantification. Only unique and razor peptides were used for quantification and a minimum count of two was required. We multiplied the LFQ intensity of the part-decell and the naïve sample by correction factors of 10.3 and 28.4 respectively. The calculation of the correction factors was based on tissue wet and dry weight information and the details are described in the Supplementary notes. The Perseus software 1.5.0.15 was used for downstream statistical analyses. Proteins were filtered by requiring at least two valid values in at least one sample condition. The corrected intensities were log2 transformed and missing values were replaced using data imputation by employing a width of 0.3 and a downshift of 1.2. |
| instruments | Thermo Scientific LTQ Orbitrap Velos |
| species | Rat, Mouse |
| massModifications | static: C+57.0215, variable: M+15.9949, P+15.9949, K+15.9949, K+178.0473, K+340.0995 |
