Extracellular Matrix (ECM) proteomics for the UCSD Female Reproductive TMC
Prepare tissue according to the following protocol:
For this protocol, use tissue that has been snap-frozen.
I. Sample Extraction and Digestion:
Lyophilized samples were weighed (~5mgs), and brought up in 50mM Tris-HCl (pH 7.4), .25% CHAPS, 25mM EDTA, 3M NaCl supplemented with 10ul/mL Halt Protease Arrest.
Approximately 100 mg of 3mm glass beads were used to mechanically agitate samples in a Bullet Blender
Vortexed @ 4°C for 20 min
Centrifuged at 18,000 x g for 20 minutes, supernatant removed
Steps b-d repeated x 2
Pellets subjected to chemical digestion with 1M hydroxylamine hydrochloride (HA) in 6M Gnd-HCl
Protein concentration of each sample determined by BCA
Proteolytic digestion was carried out according to the FASP protocol1 with 10 kDa molecular weight cutoff filters (Sartorius Vivacon 500 #VN01H02) using 30 ug of protein resulting from each sample.
Samples reduced with tris(2-carboxyethyl)phosphine), alkylated with 2-chloroacetamide, and digested with trypsin (1:100) at 37ºC for 14 Hrs. Peptides were recovered from the filter using successive washes with 0.2% formic acid.
Resulting peptides were analyzed by LC-MS/MS using a Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) coupled to an EASY-nLC 1200 (Thermo Fisher Scientific) operating in data dependent acquisition mode.
Raw spectra were interpreted against the human proteome database using the MSFragger-based FragPipe computational platform2
Sample preparation for LC-MS/MS
Lyophilized samples were weighed (~5mgs) and brought up in 50mM Tris-HCl (pH 7.4), .25% CHAPS, 25mM EDTA, 3M NaCl supplemented with 10ul/mL Halt Protease Arrest. Approximately 100 mg of 3mm glass beads were used to mechanically agitate samples in a Bullet Blender (NextAdvance) prior to all cellular and ECM extraction steps. Protein concentration of each fraction for each sample was measured using A660 Protein Assay (Pierce™). Proteolytic digestion was carried out according to the FASP protocol1 with 10 kDa molecular weight cutoff filters (Sartorius Vivacon 500 #VN01H02) using 30 ug of protein resulting from each fraction. Samples reduced with tris(2-carboxyethyl)phosphine), alkylated with 2-chloroacetamide, and digested with trypsin (1:100) at 37ºC for 14 hrs. Peptides were recovered from the filter using successive washes with 0.2% formic acid. Peptides were recovered from the filter using successive washes with 0.2% formic acid.
Global proteomics for all comparative method testing was carried out on a Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) coupled to an EASY-nLC 1200 (Thermo Fisher Scientific) through a nanoelectrospray LC − MS interface. Eight μL of each sample was injected into a 20 μL loop using the autosampler. The analytical column was then switched on-line at 400 nl/min over an in house-made 100 μm i.d. × 150 mm fused silica capillary packed with 2.7 μm CORTECS C18 resin (Waters; Milford, MA). LC mobile phase solvents consisted of 0.1% formic acid in water (Buffer A) and 0.1% formic acid in 80% acetonitrile (Buffer B, Optima™ LC/MS, Fisher Scientific, Pittsburgh, PA). After 22 uL of sample loading at a maximum column pressure of 700 bar, each sample was separated on a 120-min gradient at a constant flow rate of 400 nL/min. The separation gradient for cell fractions consisted of 6% buffer B from 0 to 3 minutes, followed by a linear gradient from 6 to 42% buffer B from 3 minutes to 105 minutes. Linear gradients from 6 to 36% and 6 to 24% buffer B were utilized from 3 to 105 minutes for the sECM and iECM fractions, respectively. Gradient elution was followed by a linear increase to 55% buffer B from 105 to 110 minutes and further to 95% buffer B from 110 to 111 minutes. Flow at 95% buffer B was maintained from 111 minutes to 120 minutes to remove remaining peptides. Data acquisition was performed using the instrument supplied Xcalibur™ (version 4.5) software. The mass spectrometer was operated in the positive ion mode. Each survey scan of m/z 375–1600 was followed by higher energy collisional dissociation (HCD) MS/MS (30% collision energy) using the standard AGC target and a 35 ms maximum injection time with an isolation width of 1.6 m/z. The orbitrap was used for MS1 and MS2 detection at resolutions of 120,000 and 50,000, respectively. Dynamic exclusion was performed after fragmenting a precursor 1 time for a duration of 45 sec. Singly charged ions were excluded from HCD selection.
Fragmentation spectra were searched against the UniProt humanproteome database using the MSFragger-based FragPipe computational platform2. Contaminants and reverse decoys and were added to the database automatically. The precursor-ion mass tolerance and fragment-ion mass tolerance were set to 10 ppm and .2 Da, respectively. Precursor tolerance was set to ±10 ppm and fragment tolerance was set to ±0.04 Da, allowing for 2 missed cleavages. Fixed modifications were set as carbamidomethyl (C). Variable modifications were set as oxidation (M), oxidation (P) (hydroxyproline), Gln->pyro-Glu (N-term Q), deamidated (NQ), and acetyl (Protein N-term). Results were filtered to 1% FDR at the peptide and protein level.
1Wiśniewski, J.R., 2016. Quantitative evaluation of filter aided sample preparation (FASP) and multienzyme digestion FASP protocols. Analytical chemistry, 88(10), pp.5438-5443.
2Kong, A.T., Leprevost, F.V., Avtonomov, D.M., Mellacheruvu, D. and Nesvizhskii, A.I., 2017. MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry–based proteomics. Nature methods, 14(5), pp.513-520.