A Proteomic Analysis of Human Hemodialysis Fluid

G. Hanumanthu Reddy,Paul J. Scheel,Jakob Bunkenborg,Henrik Molina,Babylakshmi Muthusamy,Akhilesh Pandey

doi:10.1074/mcp.m500042-mcp200

Abstract

The vascular compartment is an easily accessible compartment that provides an opportunity to measure analytes for diagnostic, prognostic, or therapeutic indications. Both serum and plasma have been analyzed extensively by proteomic approaches in an effort to catalog all proteins and polypeptides. Limitations of such approaches in obtaining a comprehensive catalog of proteins include the fact that a handful of proteins constitute over 90% of plasma protein content and that the renal glomeruli filter out proteins and polypeptides that are smaller than 66 kDa from blood. We chose to study hemodialysis fluid because it contains a higher concentration of small proteins and polypeptides and is also simultaneously depleted of the most abundant proteins present in blood. Using gel electrophoresis in combination with LC-MS/MS, we identified 292 proteins of which greater than 70% had not been previously identified from serum or plasma. More than half of the proteins identified from the hemodialysis fluid were smaller than 40 kDa. We also found 50 N-terminally acetylated peptides that allowed us to unambiguously map the N termini of mature forms of the corresponding proteins. Several identified proteins, including cytokines, were only present as predicted transcripts in data bases and thus represent novel proteins. The proteins identified in this study could serve as biomarkers in serum using more sensitive methods such as ELISA-specific antibodies.

Highlights

The vascular compartment is an accessible compartment that provides an opportunity to measure analytes for diagnostic, prognostic, or therapeutic indications
Hemodialysis fluid has previously been used as a source of polypeptides [6] due to its higher concentration of low molecular weight components, but no comprehensive list of constituents present in the hemofiltrate has yet been published
Our strategy involved one-dimensional gel electrophoresis separation and in-gel digestion of proteins followed by LCMS/MS for identifying proteins in the hemodialysis fluid

Summary

EXPERIMENTAL PROCEDURES

The gel pieces were shrunk using 100% acetonitrile, and proteins were reduced by addition of 0.1 M dithiothreitol followed by an incubation step at 56 °C for 45 min. After an additional wash and shrinkage, 10 ng/␮l trypsin in 0.1 M NH4HCO3 sufficient to cover the gel pieces was added followed by an incubation on ice for 20 min. An extraction step was carried out to recover the peptides from the gel slices by adding 50% acetonitrile and incubating at room temperature for 30 min. 20 ␮l of serum were diluted to 100 ␮l in Buffer A (Buffer A and Buffer B as supplied in the multiple affinity removal kit by Agilent Technologies) prior to loading onto the column at 250 ␮l/min in Buffer A. Vides the MS/MS spectra of all proteins that were identified on the basis of a single peptide

RESULTS AND DISCUSSION

99 Glutathione transferase

43 Chromosome 6 open reading frame 115

Conclusions