Sample Prefractionation
1| 50 μl human vitreous contains approximately 50-150 μg of protein, as determined by the Bio-Rad protein assay method. Solubilize samples in 2X sample buffer and heat at 96°C for 5 min. Place samples briefly on ice until ready for use and centrifuge at 14000 rpm for 5 min in an Eppendorf centrifuge prior to loading onto SDS-PAGE. Normalize samples to undiluted vitreous volume (eg, 50 μl) for comparisons.
PAUSE POINT The treated sample can be stored at -80°C for 6 month for future runs.

2| Separate vitreous samples by SDS-PAGE
CRITICAL STEP The glass plates and all chambers and casting systems are washed thoroughly and handed with clean gloves, to avoid contamination with other proteins and keratins.

Prepare the resolving gel (12% or 10%, approx. 35 ml for one 14 ×16 cm gel) containing the following components:
Resolving gel 12%
30 % (w/v) acrylamide/ Bis 14 ml
1.5 M Tris/HCl pH 8.8, 8.75 ml
10 % (w/ v) SDS 0.35 ml
distilled water 11.73 ml

The corresponding stacking gel (4%, approx. 12.5 ml for one gels) contains:
30 % (w/v) acrylamide/ Bis 1.65 ml
0.5 M Tris/ HCl pH 6.8 3.15 ml
10 % (w/ v) SDS 0.125 ml
distilled water 7.5 ml

!CAUTION: Acrylamide and bisacrylamide are highly neurotoxic. When handling these chemicals, wear gloves and use a pipetting aid.

Load samples and prestained molecular weight markers on gel.
Carry out electrophoresis at a constant current of 30 mA per gel (1.5-mm-thick). Under these conditions, the gel will take approximately 3–4 h to run. If it is more convenient to run the gel for a longer period (e.g. 8 h), reduce the current to 15 mA and reduce the current to 7 mA per gel for overnight. Overnight running improves resolution and is preferred for the sample of more than 100 μg protein. When the tracking dye reaches the bottom of the gel, turn the power supply off and disconnect the power cables.

Coomassie Brilliant Blue staining
3| Submerge the gel in enough Coomassie Blue staining solution that the gel floats freely in the tray. Shake slowly on a laboratory shaker or rocker for 30-60 min.
!CAUTION: Covered plastic trays work well and minimize exposure to methanol and acetic acid vapors.

4| Destain the gel with several changes of destaining solution until the background is transparent. (Gentle rocking for about 2-3 hours).
CRITICAL STEP Destaining must be monitored visually and adjusted accordingly.

5| Acquire gel images using your chosen imaging system.

6| Gel can be stored in 7% acetic acid solution at 4°C for several weeks prior to in-gel digestion, but stopping at next step is preferred.

7| Divide the entire lane for each sample into 60-70 slices of 1 mm width and place in eppendorf tubes. The slices can be stored at -20°C for several weeks prior to in-gel digestion.
PAUSE POINT Gels can be stored in 7% acetic acid solution at 4°C for several weeks without significant loss of protein. For long-term storage, protein slices to be identified by MS should be cut out and frozen at –80ºC.

In gel digestion
CRITICAL STEP Wear gloves throughout this process. Thoroughly clean all equipment and use disposable components when possible.

8| Add 1 ml of water to remove any residual acid. Remove and discard water.

9| Add 1 ml of a 50% acetonitrile: 50% 50 mM ammonium bicarbonate (from the 100 mM stock solution) and soak for 30 min to 1 h. If coomassie stain is very dark, soak for at least 1 h, change the 1 ml of a 50% acetonitrile: 50% 50 mM ammonium bicarbonate if necessary.

10| Remove 1 ml of a 50% acetonitrile: 50% 50 mM ammonium bicarbonate.

11| Add 200 μl of acetonitrile to completely dehydrate gel slices (acrylamide slices should turn opaque white). Let sit in acetonitrile for approximately 10 min and then remove acetonitrile.

12| Dry gel chips in the speed vac (10-15 min – until completely dry).

13| Rehydrate slices by adding 90 μl trypsin digest solution and incubate overnight at 37ºC.

Peptide extraction
14| Remove supernatant and save for analysis.

15| Rinse slice at 3 times with 80% acetonitrile, 1% formic acid.

16| Remove liquid and pool with supernatant from step 14.

17| Dry down digest in Speed Vac until approximately 2 μl remains.
CRITICAL STEP Don’t over dry the digest.

18| Resuspend in 18 μl 2% Acetonitrile.
PAUSE POINT The digest can be stored at –80ºC for months.

Nano-LC-MS/MS analysis
19| Separate the samples with microcapillary reverse-phase column in line with a LTQ linear ion trap mass spectrometer. Data acquisition parameters were full scan MS (range 370 to 2000 m/z) followed by 10 data-dependent MS/MS events.
MS2 Isolation width: 2.0
MS2 Normalized Collision Energy: 25
MS2 Activation Q: 0.25
MS2 Activation Time: 30.0
Exclusion Mass Width by Mass
Exclusion Mass Width Low: 1.00
Exclusion Mass Width High: 1.50
Dynamic Exclusion Enabled
Repeat Count: 2
Repeat Duration: 30.0
Exclusion List Size: 500
Exclusion Duration: 90.0

Data analysis
20| Prepare a protein database in FASTA format. Protein databases in FASTA format are available for various species from the National Center for Biotechnology Information (NCBI)( ftp://ftp.ncbi.nih.gov/blast/db/) or from European Bioinformatics Institute (ftp://ftp.ebi.ac.uk/pub/databases/IPI/current/).

21| Index the Database with Database Manager. Indexer Parameters are
Monoisotopic
Trypsin (KR)
Fully enzymatic –cleaves at both ends
MW Range: 300-3500
Missed cleavage sites: 0
Differential/PTM: M+16 and C+71

22| Process multiple .raw files that are contained in an Xcalibur Sequence (.sld) file through TurboSEQUEST. TurboSEQUEST Search Parameters are as follows.
MW Range 600-3500
Threshold: Absolute, 5000
Precursor ion tolerance:1.4
Mass Units:AMU
Group Scan: 1
Minimun group count: 1
Minimum ion count: 15
Use Charge State: Auto
MSn level: Auto

23| Export the TurboSEQUEST search results into XML, excel or SQT format.

24| Enter resultant matches and compile into a MySQL relational database and perform proteomics computational analyses. We use an in-house program based on PHP-MySQL-Apache platform.
Data processing proceeds in five steps: parsing in to database, peptide level filtering, summarizing, protein level filtering, and reporting.
The first step is carried out by parsing the Sequest search result into a MySQL database.
The second step removes low-scoring peptide. If a peptide does not meet the following criteria, it will be removed. Cross-correlation score > 1.5, 2.0 and 2.5 for charge states +1, +2 and +3, respectively; Delta Correlation >0.1; Primary Score >200; Ranking of the Primary Score <3; and percent fragment ions >30%.
Step 3 is to combine all search results from a sample (generated from entire lane of gel slices) into a single file based on the protein accession number. Slice number information should be retained in this file for later filtering and analysis.
The step 4 is filtering the redundant proteins, trypsin, keratin, and proteins that do not have 2 unique peptides identified from a single slice or adjacent slices.
The last step is compiling the all the sample into one file. If multiple accession numbers are assigned for a peptide match then select uniform accession numbers that is consistent with all peptide matches for that protein to enable the comparison of proteins identified from different samples. The number of unique peptides identified for a given protein can be used as a semi-quantitative measure of that protein abundance among different samples.