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Phosphoproteomics of Human Urinary Exosomes
Author(s) -
Gonzales Patricia A,
Pisitkun Trairak,
Hoffert Jason,
Star Robert A,
Wang Nam Sun,
Knepper Mark A
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1158.24
Subject(s) - microvesicles , phosphorylation , phosphoproteomics , proteome , exosome , proteomics , phosphoprotein , chemistry , biology , biochemistry , protein phosphorylation , microbiology and biotechnology , protein kinase a , microrna , gene
Urinary exosomes are the low‐density membrane vesicles that originate from the internal vesicles of multivesicular bodies and are secreted into urine by every renal tubule cell types as well as glomerular podocytes. Urinary exosomes are known to contain an extensive repertoire of proteins including many potential biomarkers. These vesicles are isolated and purified by differential centrifugation from urine. In this study, we sought to define the phosphoproteome of human urinary exosomes using a combination of phosphopeptide enrichment by immobilized metal affinity chromatography and phosphorylation site identification by LC‐MS n neutral loss scanning. Nineteen phosphorylation sites corresponding to fourteen unique phosphoproteins were identified in exosomal proteins from human urine samples. The identifications include phosphorylation sites previously identified in AQP2 (S256), NKCC2 (T118 and S120), CHMP2B (S199), HSP90AB1 (S255) and SPP1 (S192, S197, and S207). Novel phosphorylation sites were identified on the following proteins: GPRC5B, GPRC5C, RRAS2, VPS4B, cytochrome b reductase, proteasome alpha 3, mucin 1, and the sodium chloride cotransporter (NCC). NCC was phosphorylated at S811. We conclude that human urinary exosomes contain many phosphoproteins. This study demonstrates the potential for identification of novel phosphoproteins from human urinary exosomes using shotgun phosphoproteomic analysis.