In-solution Digestion of ECM-Enriched Proteins Samples for Mass Spectrometry Analysis
Ikram Isa, Alexandra Naba, jconsi
Abstract
The pellet obtained after the decellularization procedure and removal of SDS is highly enriched in insoluble ECM proteins. For further analysis by mass spectrometry, these proteins need to be digested into peptides. Note that, as a consequence of ECM protein insolubility, it is not possible at this step to measure the protein concentration of the sample. We thus provide volumes of reagents to digest the ECM-enriched samples into peptides based on the size (mm) or dry weight of the ECM-enriched pellet (Table 1).
Before start
The solutions of ammonium bicarbonate, urea, DTT, iodoacetamide and trifluoroacetic acid all need to be freshly prepared. * We recommend using low retention microcentrifuge tubes and tips to maximize recovery
Steps
Protein resuspension and reduction
Resuspend the ECM-enriched sample by adding the appropriate volume of 8 M urea urea ( urea is dissolved in 100 mM ammonium bicarbonate ) to the ECM-enriched pellet and add Dithiothreitol(DTT) DTT) at a final concentration of 10 mM (see Table 1). Incubate with continuous agitation at 1400rpm for 2h 0m 0s at37°C.
Protein alkylation
Protein alkylation
Prepare a 250mM iodoacetamide solution in 100 mM ammonium bicarbonate .
Cool the sample to Room temperatureand add the iodoacetamide to a final concentration of 25 mM . For complete alkylation, the DTT:iodoacetamide ratio should be between 1:2.5 and 1:3.
Incubate in the dark for 0h 30m 0s at Room temperature.
Protein deglycosylation
Deglycosylation is needed to remove carbohydrate side chains that interfere with identification of peptides modified by N-linked glycosylation.
Dilute to 2 M urea urea with 100 mM ammonium bicarbonate and add the appropriate amount of PNGaseF (see Table 1). Incubate with continuous agitation at 1400rpm for 2h 0m 0s at 37°C.
Digestion - Day 1
Digestion - Day 1
Add Lys-C (refer to Table 1 for volume amounts) Lys-C (refer to Table 1 for volume amounts) and incubate with continuous agitation at 1400rpm for 2h 0m 0sat 37°C.
Add trypsin trypsin (refer to Table 1 for volume amounts) and incubate with continuous agitation at 1400rpm 2h 0m 0s at 37°C
Digestion - Day 2
Digestion - Day 2
Add a second aliquot of trypsin trypsin (refer to Table 1 for volume amounts) to the sample and incubate with continuous agitation at 1400rpm for an additional2h 0m 0s at 37°C.
Acidification
Acidification
Upon completion of the digestion, the trypsin is inactivated by acidifying the sample with freshly prepared 50% trifluoro-acetic acid (TFA) in HPLC-grade water . The sample should reach <2
Centrifuge the acidified sample at 16000x g,0h 0m 0s for 0h 5m 0sat Room temperature. Collect the supernatant in a clean low-retention tube. At this point, the peptide solution can be stored at -20°C prior to desalting:
Desalting of Peptides to Prepare for Mass Spectrometry Analysis.
