Proteome Patterns in Uremic Plasma

Abstract

In patients with chronic kidney disease (CKD), peptides and proteins circulate at altered concentrations versus in healthy individuals. We have characterized proteome samples from 7 pooled CKD stage 5 patients not yet on dialysis and with no known co-morbidities. We also analyzed pooled plasma samples from 7 healthy age- and sex-matched controls. After immunodepletion of the 6 most abundant plasma proteins, HPLC and SDS-PAGE patterns differed between the healthy and disease groups. The differing proteins were identified by peptide mass fingerprinting using MALDI mass spectrometry and verified with electrospray tandem mass spectrometry sequence analysis. Multiple differences in at least 19 HPLC fractions were observed, from which we identified 29 proteins, 25 in greater yield and 4 in lower yield than in the healthy controls, adding at least 6 protein components to those that were previously known to be altered in CKD.

Differential hemoglobin A sequestration between hemodialysis modalities

“…The results cover 50 protein spots identified. However, the majority of these identifications (40 proteins) were the same as proteins detected in our previous correlation of protein differences with chronic kidney disease (CKD) (7,8) and are not reported again. The same correlation may apply to the 10 additional proteins now identified, but as some may also reflect modality correlations, all novel identifications are given in the Supplement (Supplementary Table S1), although difficult to interpret because of protein multiplicity in some fractions.…”

“…The plasma samples were immunodepleted for albumin, IgG, antitrypsin, IgA, transferrin and haptoglobin on a multiple affinity removal system (MARS) column (Agilent Technologies, Santa Clara, CA, USA), as described (7,8) by application of 80 μl plasma after dilution in a neutral buffer, pH 7.4 and elution at 0.5 ml/min. The flow through fraction was pooled from 12 runs, buffer-exchanged in a spin concentrator with 10 kDa cutoff (Amicon Ultra, Millipore, Billerica, MA, USA) to 50 mM Tris-HCl, pH 7.4 and then stored at −80°C until analysis.…”

“…Bands were excised manually and digested with trypsin in a Mass PREP robotic protein-handling system (Waters, Milford, CT, USA) after destaining, DTT reduction and iodoacetamide alkylation (7,8). After extraction and concentration, the material was applied to a MALDI target plate in a 1:1 ratio (v/v) with α-cyano 4-hydroxycinnamic acid (saturated in 60% acetonitrile/0.1% TFA).…”

Kidney Disease

“…Anorexia and nutritional status may be improved by normalization of plasma branched -chain amino acids through branched - none of the currently favored biomarkers have been demonstrated to accurately refl ect nutritional status in CKD (Ikizler et al , 1999 ;Stenvinkel et al , 2002 ;de Mutsert et al , 2009 ). Instead, strong associations between several biomarkers and mortality seem to derive, at least in part, from their close association with infl ammation (de Mutsert et al , 2008 ), and in part from the important role of the kidney in metabolizing circulating peptides (Naseeb et al , 2008 ). However, as they are commonly used, we will shortly review the most common biomarkers: plasma concentrations of albumin, pre -albumin, and transferrin (Locatelli et al , 2002 ;Kamimura et al , 2005 ).…”

Update of uremic toxin research by mass spectrometry

“…They also found 50 N-terminally acetylated peptides. Naseeb et al (2008) analyzed uremic plasma from stage-5 CKD patients not yet on dialysis, and from normal plasma. After immunodepletion of the six most-abundant plasma proteins, they used HPLC and SDS-PAGE to differentiate protein patterns between the normal and uremic groups.…”