Standardized Approach to Proteome Profiling of Human Serum Based on Magnetic Bead Separation and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Baumann, Sven; Ceglarek, Uta; Fiedler, Georg Martin; Lembcke, Jan; Leichtle, Alexander Benedikt; Thiery, Joachim

doi:10.1373/clinchem.2004.047308

Cited by 208 publications

(135 citation statements)

References 29 publications

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“…Despite this, proteomic coverage is still limited and few if any robust cancer biomarkers have been identified using classical proteomic profiling methods. The need for identical handling of clinical samples is also of paramount importance in order to avoid largely proteolysis‐driven changes unrelated to biological variation and has been highlighted by numerous studies 12, 13, 14, 15, 16, 17. Finally, issues related to reproducibility and the robustness of class‐discriminating algorithms used for proteomic biomarker discovery have also been raised 18, 19.…”

Section: Introductionmentioning

confidence: 99%

Discovery of serum biomarkers of ovarian cancer using complementary proteomic profiling strategies

Timms

Arslan-Low

Kabir

et al. 2014

Proteomics Clinical Apps

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PurposeOvarian cancer is a devastating disease and biomarkers for its early diagnosis are urgently required. Serum may be a valuable source of biomarkers that may be revealed by proteomic profiling. Herein, complementary serum protein profiling strategies were employed for discovery of biomarkers that could discriminate cases of malignant and benign ovarian cancer.Experimental designIdentically collected and processed serum samples from 22 cases of invasive epithelial ovarian cancer, 45 benign ovarian neoplasms, and 64 healthy volunteers were subjected to immunodepletion and protein equalization coupled to 2D‐DIGE/MS and multidimensional fractionation coupled to SELDI‐TOF profiling with MS/MS for protein identification. Selected candidates were verified by ELISA in samples from malignant (n = 70) and benign (n = 89) cases and combined marker panels tested against serum CA125.ResultsBoth profiling platforms were complementary in identifying biomarker candidates, four of which (A1AT, SLPI, APOA4, VDBP) significantly discriminated malignant from benign cases. However, no combination of markers was as good as CA125 for diagnostic accuracy. SLPI was further tested as an early marker using prediagnosis serum samples. While it rose in cases toward diagnosis, it did not discriminate prediagnosis cases from controls.Conclusions and clinical relevanceThe candidate biomarkers warrant further validation in independent sample sets.

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Section: Introductionmentioning

confidence: 99%

Discovery of serum biomarkers of ovarian cancer using complementary proteomic profiling strategies

Timms

Arslan-Low

Kabir

et al. 2014

Proteomics Clinical Apps

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“…Many current diagnostics rely on the measurement of highly characterized, stable protein or peptide biomarkers. In a number of current studies, blood is expected to be an enormous resource of new protein or peptide biomarkers, but many of these are expected to be either potentially labile or in low abundance, complicated by the tremendous dynamic range of blood protein concentration masking this valuable, dilute content (1,3,4).…”

Section: Introductionmentioning

confidence: 99%

Thrombin induces broad spectrum proteolysis in human serum samples

O'Mullan¹,

Craft²,

Yi³

et al. 2009

Clinical Chemistry and Laboratory Medicine

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Background: During clotting, a thrombin cleaves fibrinogen releasing fibrinopeptide A (FPA). FPA is easily identified in serum using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Using MALDI-TOF MS, we observed multiple, progressively shorter fragments of serum FPA. Following ambient incubation of serum, variations in the content of FPA fragments occur over time. Denaturation of a thrombin by heating the serum sample appears to minimize this variation. These observations suggest that intrinsic proteolytic and peptidolytic activity is elevated in serum and perhaps originates from the coagulation cascade enzymes themselves, especially a thrombin. Methods: Extrinsic addition of a thrombin to a subset (3-30 kDa) of plasma proteins was carried out to induce proteolysis and to examine the resultant peptides to reveal a thrombin susceptible parent proteins. One of these identified proteins, hemopexin, was directly digested by a thrombin and the peptides examined to confirm the observations from the initial plasma protein digestion. Results: Extrinsic addition of a thrombin to a subset (3-30 kDa) of plasma proteins results in wide-spread digestion of proteins unrelated to coagulation, revealing a substrate range encompassing more than fibrinogen. Direct digestion of one of these proteins, hemopexin, by a thrombin confirms these observations. Conclusions: The resulting peptides indicate broad tolerance beyond the consensus R-G cleavage site of fibrinogen; in fact, there appears to be no bias for the amino acid following the R/K residue. These data support our hypothesis that the enzymatic activities inherent to coagulation, or at least to thrombin, contribute to destabilization of the protein and peptide content of serum. Clin Chem Lab Med 2009;47:685-93.

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“…Recently, a detection strategy based on serum peptide profiling by magnetic beads has been developed with a distinct advantage for this situation [15]. The ability of the magnetic beads to separate and enrich peptides from serum makes it possible to detect peptide toxins [16].…”

Section: Introductionmentioning

confidence: 99%

Rapid Detection of Ricin in Serum Based on Cu-Chelated Magnetic Beads Using Mass Spectrometry

Zhao

Song

Wang

et al. 2016

J. Am. Soc. Mass Spectrom.

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Abstract. The protein toxin ricin obtained from castor bean plant (Ricinus communis) seeds is a potent biological warfare agent due to its ease of availability and acute toxicity.In this study, we demonstrated a rapid and simple method to detect ricin in serum in vitro. The ricin was mixed with serum and digested by trypsin, then all the peptides were efficiently extracted using Cu-chelated magnetic beads and were detected with matrixassisted laser desorption/ionization time-of-flight mass spectrometry. The specific ricin peptides were identified by Nanoscale Ultra Performance liquid chromatography coupled to tandem mass spectrometry according to their sequences. The assay required 2.5 hours, and a characteristic peptide could be detected down to 4 ng/μl and used as a biomarker to detect ricin in serum. The high sensitivity and simplicity of the procedure makes it valuable in clinical practice.

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Standardized Approach to Proteome Profiling of Human Serum Based on Magnetic Bead Separation and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Cited by 208 publications

References 29 publications

Discovery of serum biomarkers of ovarian cancer using complementary proteomic profiling strategies

Discovery of serum biomarkers of ovarian cancer using complementary proteomic profiling strategies

Thrombin induces broad spectrum proteolysis in human serum samples

Rapid Detection of Ricin in Serum Based on Cu-Chelated Magnetic Beads Using Mass Spectrometry

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