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Ben-Bo Gao

Xiaohong Chen

Edward P. Feener

Large-scale vitreous fluid proteomics using 1D SDS-PAGE, tandem mass spectrometry, and a relational database

Mass spectrometry-based proteomics is widely used for the de novo identification of proteins from body fluids \(1). The prefractionation of complex mixtures prior to tandem mass spectrometry can dramatically improve sensitivity for low abundance proteins. A variety of protocols involving reverse phase or ion exchange chromatography, affinity enrichment methods, and in gel electrophoresis has been utilized to prefractionate these complex mixtures. Protein fractionation by one-dimensional sodium dodecylsulphate-polyacrylamide gel electrophoresis \(1D SDS-PAGE) provides high resolution, concentrates individual proteins and their resultant tryptic peptide a limited number of fractions, provides an estimate protein molecular weight, and can be used to generate a limited number of fractions that includes the entire sample. We have used preparative 1D SDS-PAGE to fractionate individual vitreous samples into 60 to 70 fractions, processed individual fractions to isolate tryptic digests, and performed sequential tandem mass spectrometric analysis using a LTQ Linear trap mass spectrometer. We developed PHP-MySQL database based software to compile, organize, and filter the data generated from each fraction from each sample and compile results a large number of independent samples into a single table. The protocols that we have developed for proteomic data acquisition and bioinformatic analysis are described.

Bio-Rad protein assay \(Bio-Rad; cat. no. 500-0006).


Beta-mercaptoethanol \(Sigma; cat. no. M-3148)


0.5M Tris-HCL Buffer pH 6.8  \(Bio-Rad; cat. no. 161-0799)


1.5M Tris-HCL Buffer pH 8.8  \(Bio-Rad; cat. no. 161-0798)


SDS \(Sigma; cat. no. L6026-250G)


30% Acrylamide/ bisacrylamide Solution  \(Bio-Rad; cat. no. 161-0158) \! CAUTION Acrylamide and bisacrylamide are highly neurotoxic. When handling these chemicals, wear gloves and use a pipetting aid.


Tetramethylethylenediamine \(TEMED)Bio-Rad, cat. no. 161-0801


Bromophenol blue \(FisherBiotech; cat. no. BP115-25))


Glycerol \(FisherBiotech; cat. no. BP-229-4)


Precision Plus Protein Standards \(Bio-Rad; cat. no. 161-0363)


Coomassie Brilliant Blue R-250 \(Bio-Rad; cat. no. 161-0400)


HPLC-grade water \(J.T.Baker; cat. no. 4218-02)


HPLC-grade acetonitrile \(ACN) \(J.T.Baker; cat. no. 9017-02) \! CAUTION  ACN is toxic. When handling, wear gloves and use a pipetting aid.


HPLC-grade formic acid \(J.T.Baker; cat. no. 0129-01)


Ammonium bicarbonate \(J.T.Baker; cat. no. 3003-01)


Sequencing-grade trypsin \(Promega; cat. no. V5111).


Trifluoroacetic acid \(TFA) \(Sigma; cat. no. 431788-100G) \! CAUTION  TFA is toxic. When handling, wear gloves and use a pipetting aid. CRITICAL STEP use glass pipette \(Fisher; 2ml).





**REAGENT SETUP**


2 x sample buffer: 1.26 ml 1.5M Tris Tris-HCl \(62.5 mM) pH 6.8, 5 ml 50% glycerol \(25%), 0.2g SDS \(2%), 1 mg Bromophenol Blue \(0.01%), adjust to 10 ml with distilled water. Store in 475 &#x3BC;l aliquots at -20 &#xB0;C.  Add 25 &#x3BC;l of b-mercaptoethanol per 475 &#x3BC;l of sample buffer for a final concentration of 5% beta-mercaptoethanol before use. As an alternative, dithiothreitol \(DTT) may be used at a final concentration of 350 mM \(54 mg/ml).


10% SDS stock solution: 10 g SDS Water to 100 ml. Store at room temperature.


10X SDS PAGE gel running buffer:  60.55 g Tris Base, 288.27 g glycine, 20 g SDS Water to 2 L. Store at room temperature.


10% Ammonium Persulfate \(APS): 1.0g APS Water to 10 ml Store at 4&#xB0;C. \(Prepare fresh daily).


Coomassie brilliant blue Stain solution:  40% methanol / 10% acetic acid/ 0.025% Coomassie Brilliant Blue R-250. Filtered through Whatman #1 paper.


Destaining solution: 40% methanol / 10% acetic acid/


Trypsin digest solution \(4.4 ng/&#x3BC;l): 20 &#x3BC;g trypsin \(1 vial) + 4.5 ml of 40 mM Ammoniumbicarbonate in 9% Acetonitrile


HPLC Solution A: 0.1% formic acid \(v/v) in water


HPLC Solution B: 0.1% formic acid \(v/v)in acetonitrile

LTQ 2-dimensional linear ion trap mass spectrometer \(Thermo Electron) equiped with a nanospray ionization source and Aquasil C18 reversed-phase nano-column \(particle size 5 um,  pore size 100 &#xC5;, 75 mm inner diameter, 15um tip) \(New Objective; cat. no. PFC7515-AQ-10-3PK)





LC PACKINGS HPLC system, consisting of a FAMOS Microautosampler, Switchos II: advanced microcolumn switching unit, nano-precolumn: C18 PepMap100, 5um, 100 &#xC5;, 300 um i.d. 1mm, and Ultimate Nano HPLC.





Electrophoresis power supply \(EPS 60, Amersham Pharmacia Biotech)


MultiTemp III Thermostatic Circulator \(Pharmacia Biotech)


SE 600 Standard Dual Cooled Vertical Electrophoresis Unit with spacers \(1.5-mm), combs, and glass plates \(Pharmacia Biotech)





Rotary laboratory shaker


DNA120 SpeedVac \(Thermo Savant) 


Centrifuge 5417C \(Eppendorf) 





SOFTWARE TOOLS


BioWorks 3.2 software \(Thermo Electron).


PHP 5.2.0 \(http://www.php.net/)  


Apache \(http://www.apache.org/)


MySQL \(http://www.mysql.com/)





EQUIPMENT SETUP


Nano-LC-MS/MS utilzes the following HPLC gradient of buffer A and buffer B.





Time interval  Gradient


0&#x2013;4 min    2&#x2013;2% B


4&#x2013;50 min  2&#x2013;98% B


50-55 min  98-98% B


55&#x2013;55.1 min  98-2% B


55.1&#x2013;60 min  2&#x2013;2% B





Injection Volume: 6 &#x3BC;l, 


Flow rate: 200 nl/min.





Parameters of LTQ: 


Scan range: 370-2000. The mass spectrum is recorded from 0 min to 60 min using a sequence of 1 MS following 10 data dependent  MS/MS events.

**Sample Prefractionation**


1| 50 &#x3BC;l human vitreous contains approximately 50-150 &#x3BC;g of protein, as determined by the Bio-Rad protein assay method. Solubilize samples in 2X sample buffer and heat at 96&#xB0;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 &#x3BC;l) for comparisons.


PAUSE POINT The treated sample can be stored at -80&#xB0;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 &#xD7;&#xF020;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&#x2013;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 &#x3BC;g 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&#xB0;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&#xB0;C for several weeks prior to in-gel digestion. 


PAUSE POINT Gels can be stored in 7% acetic acid solution at 4&#xB0;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 &#x2013;80&#xBA;C.





**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 &#x3BC;l 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 &#x2013; until completely dry).





13| Rehydrate slices by adding 90 &#x3BC;l trypsin digest solution and incubate overnight at 37&#xBA;C.  





**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 &#x3BC;l remains.


CRITICAL STEP Don&#x2019;t over dry the digest.





18| Resuspend in 18 &#x3BC;l 2% Acetonitrile.


PAUSE POINT  The digest can be stored at &#x2013;80&#xBA;C 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 &#x2013;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 &gt; 1.5, 2.0 and 2.5 for charge states +1, +2 and +3, respectively; Delta Correlation &gt;0.1; Primary Score &gt;200; Ranking of the Primary Score &lt;3; and percent fragment ions &gt;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.

SDS-PAGE prefractionation: 1-2 days


Gel staining with Coomassie Brilliant Blue: 0.5-1 days 


In gel trypsin digestion: 4-6 h per lane


Peptide extraction 4-6 h per lane 


Nano-LC-MS/MS analysis: 60 h per lane


Data analysis: 60 h per lane

This method identifies approximately 50 to 200 unique proteins per vitreous sample.

1.  Zhang,Y. _et al._ MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes. _Nucleic Acids Res._ \(2006).

National Institutes of Health \(grants DK 60165, DK 36836), Juvenile Diabetes Research Foundation \(1-2005-1047), Massachusetts Lions Eye Research Fund, and Adler Foundation.

An open repository of community-contributed protocols sponsored by Nature Research.

Learn more about Protocol Exchange

Method Article

Ben-Bo Gao, Xiaohong Chen, Edward P. Feener

Ben-Bo Gao

Joslin Diabetes Center

Xiaohong Chen

Joslin Diabetes Center

Edward P. Feener

Joslin Diabetes Center

DOI:

10.1038/nprot.2007.161

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Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation

by Gao, B.B. et al.

Nature Medicine (29 December, 2006)

An open repository of community-contributed protocols sponsored by Nature Research.

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Method Article

Ben-Bo Gao, Xiaohong Chen, Edward P. Feener

Ben-Bo Gao

Joslin Diabetes Center

Xiaohong Chen

Joslin Diabetes Center

Edward P. Feener

Joslin Diabetes Center

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DOI:

10.1038/nprot.2007.161

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