Limits...
Technical Brief: a novel strategy for enrichment of trabecular meshwork protease proteome.

Picciani R, Junk AK, Bhattacharya SK - Mol. Vis. (2008)

Bottom Line: The resultant protein solution is precipitated with acetone, fractionated on SDS-PAGE, in situ trypsin digested, and subjected to mass spectrometry.This relatively simple protocol enables improved capture of cytosolic proteases.Both serine and cysteine proteases were captured using this strategy with improved coverage compared to our previous identification without affinity enrichment.

View Article: PubMed Central - PubMed

Affiliation: Bascom Palmer Eye Institute, University of Miami, Miami, FL 33136, USA.

ABSTRACT
We present a novel and simple enrichment strategy to capture trabecular meshwork (TM) protease proteome. The method relies on fractionation of TM tissue into cytosolic and nuclear extracts and subsequent affinity enrichment of proteases on peptide inhibitors. A large repertoire of available protease substrate analog peptides enables an improved capture of TM protease proteome compared to SDS-PAGE fractionation alone. Peptide analog inhibitors of protease substrates are immobilized on a protein A or G column using 254 nm intense ultraviolet (UV) light. The TM cytosolic protein extract incubated on the column is eluted with salt or a buffer with a low hydrogen ion concentration. The resultant protein solution is precipitated with acetone, fractionated on SDS-PAGE, in situ trypsin digested, and subjected to mass spectrometry. This relatively simple protocol enables improved capture of cytosolic proteases. We identified 20 previously reported TM proteins from a single donor tissue using affinity enrichment. The majority of identified proteins were either intracellular proteases or known protease inhibitors. Both serine and cysteine proteases were captured using this strategy with improved coverage compared to our previous identification without affinity enrichment.

Show MeSH
Effect of Initial material and time of incubation on relative recovery of proteins from immobilized affinity peptide columns. A. The relative recovery of protein was determined using fractionated Porcine cytosolic proteins on a PHAST gel using silver staining and densitometric scan of the area. The densitometric scan of product from 550 µg of initial TM extract incubated with beads for 3 h, recovered using 1 M NaCl, 100 mM KCl elution after acetone precipitation using 10 µg of carrier yeast tRNA as reference (considered as 100 percent) was used for determination of relative recovery ratios. B. Porcine TM extracts (500 µg initial load) was used for incubation at different times as indicated was eluted with 1 M NaCl and 100 mM KCl using carrier yeast tRNA. Results of three independent determinations were used to determine the mean and standard deviation shown here.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2386506&req=5

f2: Effect of Initial material and time of incubation on relative recovery of proteins from immobilized affinity peptide columns. A. The relative recovery of protein was determined using fractionated Porcine cytosolic proteins on a PHAST gel using silver staining and densitometric scan of the area. The densitometric scan of product from 550 µg of initial TM extract incubated with beads for 3 h, recovered using 1 M NaCl, 100 mM KCl elution after acetone precipitation using 10 µg of carrier yeast tRNA as reference (considered as 100 percent) was used for determination of relative recovery ratios. B. Porcine TM extracts (500 µg initial load) was used for incubation at different times as indicated was eluted with 1 M NaCl and 100 mM KCl using carrier yeast tRNA. Results of three independent determinations were used to determine the mean and standard deviation shown here.

Mentions: The method presented here is to enable the capture of the protease proteome profile from single tissue samples. This method is aimed to eventually be applied to surgical TM tissue samples. While it is possible to quantify the proteins recovered from immobilized peptide columns with established biochemical methods for large initial protein loads (milligram quantities), the limited initial protein load in actual tissue samples prohibits quantification with the current biochemical methods (i.e., Bradford, Folin-Ciocalteau, Lowry, and Amido Black). For this purpose, we have performed experiments with porcine TM tissue to reduce or limit the use of precious human tissue for method development alone. Our studies indicate that reproducible relative quantification is possible for fractionated proteins on the PHAST gel system using silver staining and densitometry, but absolute quantification is not. Using porcine TM extract and densitometric scans of silver staining, the impact of the initial material (within a range of 50–500 µg) and time of incubation (1–16 h) was found to have an effect on the relative recovery of proteins (Figure 2). The densitometric scan of the recovered products from 550 µg of initial TM extract was incubated with immobilized peptide beads for 3 h, eluted with 1 M NaCl plus 100 mM KCl followed by acetone precipitation; 10 µg of carrier yeast tRNA was used as a reference (considered as 100 percent). This reference was used for calculation of the relative recovery ratios for all other elutions. To determine the effect of incubation time on relative recovery of eluted products, an initial load of 500 µg of porcine TM and was used, and elution was performed in an identical fashion as noted above.


Technical Brief: a novel strategy for enrichment of trabecular meshwork protease proteome.

Picciani R, Junk AK, Bhattacharya SK - Mol. Vis. (2008)

Effect of Initial material and time of incubation on relative recovery of proteins from immobilized affinity peptide columns. A. The relative recovery of protein was determined using fractionated Porcine cytosolic proteins on a PHAST gel using silver staining and densitometric scan of the area. The densitometric scan of product from 550 µg of initial TM extract incubated with beads for 3 h, recovered using 1 M NaCl, 100 mM KCl elution after acetone precipitation using 10 µg of carrier yeast tRNA as reference (considered as 100 percent) was used for determination of relative recovery ratios. B. Porcine TM extracts (500 µg initial load) was used for incubation at different times as indicated was eluted with 1 M NaCl and 100 mM KCl using carrier yeast tRNA. Results of three independent determinations were used to determine the mean and standard deviation shown here.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2386506&req=5

f2: Effect of Initial material and time of incubation on relative recovery of proteins from immobilized affinity peptide columns. A. The relative recovery of protein was determined using fractionated Porcine cytosolic proteins on a PHAST gel using silver staining and densitometric scan of the area. The densitometric scan of product from 550 µg of initial TM extract incubated with beads for 3 h, recovered using 1 M NaCl, 100 mM KCl elution after acetone precipitation using 10 µg of carrier yeast tRNA as reference (considered as 100 percent) was used for determination of relative recovery ratios. B. Porcine TM extracts (500 µg initial load) was used for incubation at different times as indicated was eluted with 1 M NaCl and 100 mM KCl using carrier yeast tRNA. Results of three independent determinations were used to determine the mean and standard deviation shown here.
Mentions: The method presented here is to enable the capture of the protease proteome profile from single tissue samples. This method is aimed to eventually be applied to surgical TM tissue samples. While it is possible to quantify the proteins recovered from immobilized peptide columns with established biochemical methods for large initial protein loads (milligram quantities), the limited initial protein load in actual tissue samples prohibits quantification with the current biochemical methods (i.e., Bradford, Folin-Ciocalteau, Lowry, and Amido Black). For this purpose, we have performed experiments with porcine TM tissue to reduce or limit the use of precious human tissue for method development alone. Our studies indicate that reproducible relative quantification is possible for fractionated proteins on the PHAST gel system using silver staining and densitometry, but absolute quantification is not. Using porcine TM extract and densitometric scans of silver staining, the impact of the initial material (within a range of 50–500 µg) and time of incubation (1–16 h) was found to have an effect on the relative recovery of proteins (Figure 2). The densitometric scan of the recovered products from 550 µg of initial TM extract was incubated with immobilized peptide beads for 3 h, eluted with 1 M NaCl plus 100 mM KCl followed by acetone precipitation; 10 µg of carrier yeast tRNA was used as a reference (considered as 100 percent). This reference was used for calculation of the relative recovery ratios for all other elutions. To determine the effect of incubation time on relative recovery of eluted products, an initial load of 500 µg of porcine TM and was used, and elution was performed in an identical fashion as noted above.

Bottom Line: The resultant protein solution is precipitated with acetone, fractionated on SDS-PAGE, in situ trypsin digested, and subjected to mass spectrometry.This relatively simple protocol enables improved capture of cytosolic proteases.Both serine and cysteine proteases were captured using this strategy with improved coverage compared to our previous identification without affinity enrichment.

View Article: PubMed Central - PubMed

Affiliation: Bascom Palmer Eye Institute, University of Miami, Miami, FL 33136, USA.

ABSTRACT
We present a novel and simple enrichment strategy to capture trabecular meshwork (TM) protease proteome. The method relies on fractionation of TM tissue into cytosolic and nuclear extracts and subsequent affinity enrichment of proteases on peptide inhibitors. A large repertoire of available protease substrate analog peptides enables an improved capture of TM protease proteome compared to SDS-PAGE fractionation alone. Peptide analog inhibitors of protease substrates are immobilized on a protein A or G column using 254 nm intense ultraviolet (UV) light. The TM cytosolic protein extract incubated on the column is eluted with salt or a buffer with a low hydrogen ion concentration. The resultant protein solution is precipitated with acetone, fractionated on SDS-PAGE, in situ trypsin digested, and subjected to mass spectrometry. This relatively simple protocol enables improved capture of cytosolic proteases. We identified 20 previously reported TM proteins from a single donor tissue using affinity enrichment. The majority of identified proteins were either intracellular proteases or known protease inhibitors. Both serine and cysteine proteases were captured using this strategy with improved coverage compared to our previous identification without affinity enrichment.

Show MeSH