Technical brief

A novel strategy for enrichment of trabecular meshwork protease proteome

Renata Picciani, Anna K. Junk, Sanjoy K. Bhattacharya

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)871-877
Number of pages7
JournalMolecular Vision
Volume14
StatePublished - May 14 2008
Externally publishedYes

Fingerprint

Trabecular Meshwork
Proteome
Peptide Hydrolases
Protease Inhibitors
Peptides
Polyacrylamide Gel Electrophoresis
Proteins
Cysteine Proteases
Staphylococcal Protein A
Serine Proteases
Ultraviolet Rays
Acetone
Trypsin
Mass Spectrometry
Buffers
Salts
Tissue Donors

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Picciani, R., Junk, A. K., & Bhattacharya, S. K. (2008). Technical brief: A novel strategy for enrichment of trabecular meshwork protease proteome. Molecular Vision, 14, 871-877.

Technical brief : A novel strategy for enrichment of trabecular meshwork protease proteome. / Picciani, Renata; Junk, Anna K.; Bhattacharya, Sanjoy K.

In: Molecular Vision, Vol. 14, 14.05.2008, p. 871-877.

Research output: Contribution to journalArticle

Picciani, R, Junk, AK & Bhattacharya, SK 2008, 'Technical brief: A novel strategy for enrichment of trabecular meshwork protease proteome', Molecular Vision, vol. 14, pp. 871-877.
Picciani, Renata ; Junk, Anna K. ; Bhattacharya, Sanjoy K. / Technical brief : A novel strategy for enrichment of trabecular meshwork protease proteome. In: Molecular Vision. 2008 ; Vol. 14. pp. 871-877.
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