Frequently Used Protocols

Gel process

I. Coomassie:

  1. De-stain gel to a clear background so that bands can be easily seen.
  2. If possible, take a picture of the gel prior to excision of gel bands and submit an image along with the sample.
  3. Excise gel band(s)/spot(s) with as little excess empty gel as possible.
  4. Place the gel band(s)/spot(s) into a microcentrifuge tube with some water.
  5. Fill out the Sample Submission Form.
  6. Drop off or send samples and submission form to the facility.

II. Silver staining:

Please refer to here for silver staining protocol.

  1. Only stain the gel long enough (usually only a few minutes) to detect the bands of interest.
  2. If possible, take a picture of the gel prior to excision of gel bands and submit an image along with the sample.
  3. Excise gel band(s)/spot(s) with as little excess empty gel as possible.
  4. Place the gel band(s)/spot(s) into a microcentrifuge tube with some water.
  5. Fill out the Sample Submission Form.
  6. Drop off or send samples and submission form to the facility

III. Sypro Ruby staining:

  1. Sypro is a mass spectrometry compatible protein stain with similar sensitivity as silver.
  2. Since the stain is a florescent dye it can not be seen by the naked eye. Please submit a picture of the gel and indicate the bands or spots to be analyzed.
  3. Excise gel band(s)/spot(s) with as little excess empty gel as possible.
  4. Place the gel band(s)/spot(s) into a microcentrifuge tube with some water.
  5. Fill out the Sample Submission Form.
  6. Drop off or send samples and submission form to the facility.

In-gel digestion

Reference

  1. Shevchenko 2007 [Nature Protocols 1:2856-2860]

Buffers:

  1. 50mM ABC (NH4HC03) (pH 8.0~pH8.5, no need to adjust pH)
  2. 8 M urea in 50mM ABC
  3. Reduction buffer (1M dithiothreitol in water)
  4. Alkylation buffer (1M iodoacetamide in water)
  5. Trypsin

Method:

  1. Protein solubilized in 200µL 8 M urea, 50mM ABC (pH 8.0) spin down any insoluble pellet.
  2. Add reduction buffer to a final concentration of 10mM and incubate 30 min at 56°C or 1h at RT.
  3. Add alkylation buffer to a final concentration of 20mM and incubate 40 min at RT in dark.
  4. Dilute sample with 5 volumes 50 mM NH4HC03.
  5. Add 1 µg trypsin/20 µg sample protein and incubate overnight at room temperature (with agitation).
  6. Desalt using SPE column, and dry down, -80 until MS analysis

LC-MS

Short version:

Proteins from each sample were dissolved in 8 M urea, then reduced and alkylated. The samples were digested with trypsin. Resulting peptides were analyzed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry(HPLC-ESI-MS/MS). The acquired MS/MS spectra were searched against the human International Protein Index (IPI) protein sequence database (version 3.85) using Maxquant with the label free quantitation (LFQ) option. The false discovery rate (FDR) was set to ≤ 1% on both protein and peptide level. Quantification was performed using normalized LFQ intensity.

Long version:

8M Urea in 50mM ABC was used to reconstitute proteins. Reduction and alkylation were done by adding DTT to a final concentration of 20 mM at 56°C for 30min followed by 40mM IAA at room temperature. The solution was then diluted 5 times by adding 50mM ABC. Trypsin was added to achieve a protein-enzyme ratio of 50:1. Digestion was performed at 37°C overnight, with continuous shaking. Digested peptides were then desalted on Sep-Pak column (Waters) and dried down in SpeedVac (ThermoFisher Scientific, San Jose, CA). Dried peptides were reconstituted in 0.5% (v/v) FA.

All MS analyses were done by HPLC-ESI-MS/MS. The system consisted of an Agilent 1100 micro-HPLC system (Agilent Technologies, Santa Clara, CA, USA) coupled with an LTQ-Orbitrap mass spectrometer (ThermoFisher Scientific, San Jose, CA) equipped with a nano-electrospray interface operated in positive ion mode. The mobile phases consisted of 0.1%(v/v) FA in water as buffer A and 0.1% (v/v) FA in acetonitrile as buffer B. Peptide separation was performed on a 75μm × 100 mm analytical column packed in-house with reverse phase Magic C18AQ resins (1.9μm; 120-Å pore size; Dr. Maisch GmbH, Ammerbuch, Germany). Briefly, the sample was loaded on the column using 98% buffer A at a flow rate of 1.5µL/min for 15min. Then, a gradient from 5% to 35% buffer B (20~50% for APols depletion test) was performed in 120 min at a flow rate of ~300nL/min obtained from splitting a 20 µL/min through a restrictor. The MS method consisted of one full MS scan from 300 to 1700 m/z followed by data-dependent MS/MS scan of the 5 most intense ions, with dynamic exclusion repeat count of 2, and repeat duration of 90 s. As well, for the experiments on the Orbitrap MS the full MS was in performed in the Orbitrap analyzer with R = 60,000 defined at m/z 400, while the MS/MS analysis were performed in the LTQ. To improve the mass accuracy, all the measurements in Orbitrap mass analyzer were performed with internal recalibration (“Lock Mass”). On the Orbitrap, the charge state rejection function was enabled, with single and “unassigned” charge states rejected.

The raw files generated by the LTQ-Orbitrap were processed and analyzed using MaxQuant, Version 1.2.2.5 using the Uniprot protein fasta database (2012, July version), including commonly observed contaminants. The following parameters were used: cysteine carbamidomethylation was selected as fixed modification; methionine oxidation, protein N-terminal acetylation, and enzyme specificity was set to trypsin. Up to two missing cleavages of trypsin were allowed. Precursor ion mass tolerances were 7 ppm, and fragment ion mass tolerance was 0.8 Da for MS/MS spectra. If the identified peptide sequences from one protein were equal to or contained within another protein’s peptide set, then the proteins were grouped together and reported as one protein group. The false discovery rate (FDR) for peptide and protein was set at 1% and a minimum length of six amino acids was used for peptides identification. Quantification was performed using normalized LFQ intensity.

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