Differential polypeptide display: the search for the elusive target

Wittke, Stefan; Kaiser, Thorsten; Mischak, Harald

doi:10.1016/j.jchromb.2003.07.013

Cited by 36 publications

(37 citation statements)

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“…Approximately 50-70% of gene products are encountered as minute-abundance proteins [4]. Traditionally, for the separation of proteins and peptides, slab gel electrophoresis and especially 2-D PAGE is yet considered the methodology of choice in protein biochemistry due to its superior resolving power [1,[5][6][7][8][9] and the parallel separation of multiple samples by means of a multilane setup [10]. However, in SDS-PAGE estimation of relative molecular mass (M r ) is based on comparison with reference proteins and accuracy of determined M r is relatively poor with 65-10% [10][11][12].…”

Section: Introductionmentioning

confidence: 99%

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Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

2005

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High throughput, outstanding certainty in peptide/protein identification, exceptional resolution, and quantitative information are essential pillars in proteome research. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has proven to meet these requirements. Soft ionization techniques, such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), have paved the way for the story of success of CE-MS in the analysis of biomolecules and both approaches are subject of discussion in this article. Meanwhile, CE-MS is far away from representing a homogeneous field. Therefore the review will cover a vast area including the coupling of different modes of CE (capillary zone electrophoresis, capillary isoelectric foscusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as on-line preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) applied to compensate for restricted detection sensitivity. Special attention is given to improvements in interfacing, namely addressing nanospray and coaxial sheath liquid design. Peptide mapping, collision-induced dissociation with subsequent tandem MS, and amendments in mass accuracy of instruments improve information validity gained from MS data. With 2-D on-line coupling of liquid chromatography (LC) and CE a further topic will be discussed. A special section is dedicated to recent attempts in establishing CE-ESI-MS in proteomics, in the clinical and diagnostic field, and in the food sector.

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Section: Introductionmentioning

confidence: 99%

“…Sensitivity levels off at gel-loadings , 25 pmol due to extraction losses [15], which are probably related to the staining conditions that denature proteins [5]. General limitations for coupling 2-D PAGE with MALDI-MS are reviewed elsewhere [6].…”

Section: Introductionmentioning

confidence: 99%

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

2005

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“…Several hundred protein spots can be discerned with 2-D PAGE. However, since each single spot must be analyzed separately by MS/MS for identification, these techniques are cumbersome [2]. In addition, 2-D PAGE shows only moderate resolution and reproducibility for the smaller proteins and peptides commonly found in urine [3].…”

Section: Introductionmentioning

confidence: 99%

Pilot study of capillary electrophoresis coupled to mass spectrometry as a tool to define potential prostate cancer biomarkers in urine

et al. 2005

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We describe the use of capillary electrophoresis (CE) coupled with mass spectrometry (MS) to identify single polypeptides and patterns of polypeptides specific for prostate cancer (CaP) in human urine. Using improved sample preparation methods that enable enhanced comparability between different samples, we examined samples from 47 patients who underwent prostate biopsy. Of this group, 21 patients had benign pathology and 26 with CaP, and these were used to define potential biomarkers, which allow discrimination between these two states. In addition, CE-MS data from these 47 urine samples were compared to that of 41 young men (control) without known or suspected clinical CaP to further confirm the polypeptides indicative for CaP. Upon crossvalidation of the same samples, several polypeptides were selected that enabled correct classification of the CaP patients with 92% sensitivity and 96% specificity. We then examined an additional 474 samples from patients with renal disease enrolled in other studies and found that 14 (3%) had polypeptides suggestive of CaP possibly indicating that they harbor clinical CaP. In conclusion, this early pilot study suggests that CE-MS of urine warrants further investigation as a tool that can identify putative biomarkers for CaP.

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“…[8][9][10] A technique allowing the reproducible analysis of all polypeptides present in complex biologic samples within a short time and suitable for high throughput analysis was recently developed by our group. [11][12][13] The stable on-line coupling of capillary electrophoresis and mass spectrometry (CE-MS), together with advances in software analysis, led to the display of more than 1000 polypeptides present in individual samples, identified via their particular migration time in the CE and their actual mass. 14 All data generated from individual samples are stored in a database, allowing intraindividual comparison of the samples taken at different time points, yielding a patient-specific pattern, as well as comparison of patient groups and controls.…”

Section: Introductionmentioning

confidence: 99%

Proteomics applied to the clinical follow-up of patients after allogeneic hematopoietic stem cell transplantation

Kaiser

Kamal

Rank

et al. 2004

Blood

156

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A phase 1 diagnostic study was performed to evaluate a novel technology for clinical proteom research based on capillary electrophoresis and mass spectrometry. Urine from 40 patients after hematopoietic stem cell transplantation (HSCT; 35 allogeneic, 5 autologous) and 5 patients with sepsis was collected for a period of 100 days and analyzed. More than 1000 different polypeptides could be detected in individual samples. Polypeptide patterns excreted in the urine of patients were significantly different from those of healthy volunteers. No significant differences were detected comparing different conditioning regimens. The aim of this study was to identify polypeptide patterns functioning as early indicators of graft-versus-host disease (GVHD). Eighteen patients developed GVHD after allogeneic HSCT. Sixteen differentially excreted polypeptides formed a pattern of early GVHD markers, allowing discrimination of GVHD from patients without complications with 82% specificity and 100% sensitivity, cross-validated. Inclusion of 13 sepsis-specific polypeptides allowed us to distinguish sepsis from GVHD with a specificity of 97% and a sensitivity of 100%. Sequencing 2 prominent GVHD-indicative polypeptides led to the identification of a peptide from leukotriene A4 hydrolase and a peptide from serum albumin. The data reveal that capillary electrophoresis and mass spectrom-

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Differential polypeptide display: the search for the elusive target

Cited by 36 publications

References 100 publications

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

Pilot study of capillary electrophoresis coupled to mass spectrometry as a tool to define potential prostate cancer biomarkers in urine

Proteomics applied to the clinical follow-up of patients after allogeneic hematopoietic stem cell transplantation

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