Differential hemoglobin A sequestration between hemodialysis modalities.

This report evaluates plasma protein patterns, dialysates and protein analysis of used dialysis membranes from the same patient under hemodialysis in three separate modalities, using high-flux membranes in concentration-driven transport (HD), convection-driven hemofiltration (HF) and combined hemodialfiltration (HDF). The plasma protein changes induced by each of the three dialysis modalities showed small differences in proteins identified towards our previous plasma analyses of chronic kidney disease (CKD) patients. The used dialysate peptide concentrations likewise exhibited small differences among the modalities and varied in the same relative order as the plasma changes, with protein losses in the order HD>HDF>HF. The membrane protein deposits allowed quantification of the relative Hb removal ratios as ~1.7 for HD and ~1.2 for HDF vs. ~1.0 for HF. Hence, plasma protein alterations, dialysate peptide contents and membrane Hb deposits all identify HD as the modality with the most extensive filtration results and exemplifies the accessibility of protein analysis of used membrane filters for evaluation of dialysis efficiencies.

Complementary LC-MS/MS Proteomic Analysis of Uremic Plasma Proteins.

OBJECTIVE: To complement an earlier analysis of protein alterations in plasma from uremic versus healthy subjects by addition of further LC-MS/MS analysis to the previously used MALDI-TOF mass analyses. METHODOLOGY: Sequence identifications of tryptic peptides from SDS gel electrophoretic fractions of immunodepleted and HPLC-fractionated plasma was performed from seven chronic kidney disease stage 5 patients (age 55 ± 14 years, glomerular filtration rate 6.9 ±2.9 mL/minute/1.73 m2) and from seven matched controls. RESULTS: About twice as many proteins were increased in uremic plasma as the previously identified. The identifications included proteins that consistently complement the two identification patterns regarding separate subunits from the same protein complex. CONCLUSION: Mass spectrometric analysis is applicable to complex plasma proteomes in clinical settings. The LC-MS/MS technique, based on individual peptide sequence analyses, gives increased identifications and also demonstrates feasibility of this technique in clinical practice.

Proinsulin C-peptide interaction with protein tyrosine phosphatase 1B demonstrated with a labeling reaction.

Based on nickel-catalyzed cross-labeling where binding partners become biotinylated, we have studied molecular interactions with an N-terminally fused GGH-tag proinsulin C-peptide. Since C-peptide has been reported to influence phosphatase activity in intact cells, we employed this method to study possible binding of the peptide to protein tyrosine phosphatase 1B (PTP-1B). C-peptide was found to interact with PTP-1B (and for control, also with antibodies to C-peptide), as did also the N- and C-terminal fragments of C-peptide which have sequence similarities with PTP-1B binding proteins. The labeling data combined with enzyme activity analysis indicate a functional interaction between acidic regions of C-peptide and specific sites of PTP-1B. Results highlight the importance of possible phosphatase/C-peptide roles in diabetes, and the usefulness of the cross-labeling reaction also for acidic peptides like C-peptide.

Proteome patterns in uremic plasma.

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.

Proteins in the insulin-secreting cell line MIN6 bind the imidazoline compound BL11282.

The imidazoline BL11282 stimulates insulin release and alters islet proteomes. Subcellular fractions of MIN6 cells showed that the membrane fraction exhibited binding to BL11282 on a Biacore chip and to BL11282-labelled magnetic beads. Bound material extracted from the beads showed a approximately 50 kDa differential band upon SDS-PAGE and a weaker 100 kDa band. The former was sensitive to competitive removal by preincubation of the fraction with BL11282, then highlighting the approximately 100 kDa band. Masspectrometric analysis revealed the approximately 50 kDa band to be EF1A and the approximately 100 kDa band to be glucose regulated P94, both of interest in insulin synthesis and secretion.

Bioinformatics processing of protein and transcript profiles of normal and transformed cell lines indicates functional impairment of transcriptional regulators in buccal carcinoma.

Normal and two transformed buccal keratinocyte lines were cultured under a standardized condition to explore mechanisms of carcinogenesis and tumor marker expression at transcript and protein levels. An approach combining three bioinformatic programs allowed coupling of abundant proteins and large-scale transcript data to low-abundance transcriptional regulators. The analysis identified previously proposed and suggested novel protein biomarkers, gene ontology categories, molecular networks, and functionally impaired key regulator genes for buccal/oral carcinoma.

Proteome analysis of vernix caseosa.

Vernix caseosa (vernix) is a white creamy substance covering the skin of the fetus during the last trimester of pregnancy. The function of vernix has long been debated but no consensus has been reached. We here report a proteome analysis of vernix using two-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization mass spectrometry and liquid chromatography coupled to tandem mass spectrometry. We have identified 41 proteins, of which 25 are novel to vernix. Notably, 39% of the identified vernix proteins are components of innate immunity, and 29% have direct antimicrobial properties. These results form a substantial contribution to the knowledge of vernix composition and demonstrate that antimicrobial protection of the fetus and the newborn child is a major and important function of vernix.

Detection of phosphorylated peptides in proteomic analyses using microfluidic compact disk technology.

A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.