Strategies for revealing lower abundance proteins in two-dimensional protein maps

Ahmed, Nuzhat; Rice, Gregory E.

doi:10.1016/j.jchromb.2004.10.070

Cited by 61 publications

(45 citation statements)

References 70 publications

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“…A comparable problem is encountered in the analysis of suction blister fluid. Since disease specific biomarkers are typically present as very low abundant proteins, these biomarkers are consequently masked by the high abundant proteins [16,17]. This is why efficient removal of abundant proteins is essential prior to biomarker identification.…”

Section: Evaluation Of Different Depletion Strategies and Initial Commentioning

confidence: 99%

“…When proteomic approaches are used to analyze suction blister fluid, the dynamic range of proteins present in this body fluid is a major bottleneck as in other body fluids such as plasma [1,15]. Since many biomarkers are low abundant proteins, these biomarkers are consequently masked by the more high abundant proteins [16,17]. The efficient removal of abundant proteins in plasma proteomics is a well-explored path to reach for the lower abundant proteins.…”

Section: Introductionmentioning

confidence: 99%

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Suction blister fluid as potential body fluid for biomarker proteins

et al. 2007

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Early diagnosis is important for effective disease management. Measurement of biomarkers present at the local level of the skin could be advantageous in facilitating the diagnostic process. The analysis of the proteome of suction blister fluid, representative for the interstitial fluid of the skin, is therefore a desirable first step in the search for potential biomarkers involved in biological pathways of particular diseases. Here, we describe a global analysis of the suction blister fluid proteome as potential body fluid for biomarker proteins. The suction blister fluid proteome was compared with a serum proteome analyzed using identical protocols. By using stringent criteria allowing less than 1% false positive identifications, we were able to detect, using identical experimental conditions and amount of starting material, 401 proteins in suction blister fluid and 240 proteins in serum. As a major result of our analysis we construct a prejudiced list of 34 proteins, relatively highly and uniquely detected in suction blister fluid as compared to serum, with established and putative characteristics as biomarkers. We conclude that suction blister fluid might potentially serve as a good alternative biomarker body fluid for diseases that involve the skin.

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Section: Evaluation Of Different Depletion Strategies and Initial Commentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Suction blister fluid as potential body fluid for biomarker proteins

et al. 2007

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“…Low-abundance proteins are seldom noticed or detected on traditional 2-DE maps due to the fact that large quantities of abundant soluble proteins usually would obscure their detection (Patton, 2002;Yamada et al, 2002;Greenough et al, 2004 andAhmed andRice, 2005). Most 2-DE based proteomic studies employ a one-extract per gel approach and the majority of proteins identified in these studies are in high abundance.…”

Section: Limitations Of Gel-based Proteomic 1451 Low Abundance Promentioning

confidence: 99%

“…However, in plasma, the predicted dynamic range of proteins is up to 12 orders of magnitude (Chevalier, 2010). Analysis of individually in parts not only provides information on protein localization, but also allows detection of protein populations that are not detectable in whole cell proteomes (Ahmed and Rice, 2005). Generally, it is require to remove abundant proteins from the sample to detect the low-abundance proteins.…”

Section: Limitations Of Gel-based Proteomic 1451 Low Abundance Promentioning

confidence: 99%

Biochemical and proteomic investigation of bovine nasal secretion

Ghazali

Jonsson

Burchmore

et al. 2012

Farm Animal Proteomics

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“…Nuclear protein extracts are widely employed to study DNA binding of TFs in electrophoretic mobility shift assays or to study trafficking of TFs between the cytosol and the nucleus. Nuclear protein extracts are also wellsuited for nuclear proteome analysis as subcellular fractionation greatly reduces the complexity of the protein mixture, thus allowing detection of the low abundant TFs [1,2,12]. Preparation of nuclear protein extracts from fresh tissue samples or cultured cells is relatively straightforward.…”

Section: Introductionmentioning

confidence: 99%

Nuclear protein extraction from frozen porcine myocardium

et al. 2010

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Protocols for the extraction of nuclear proteins have been developed for cultured cells and fresh tissue, but sometimes only frozen tissue is available. We have optimized the homogenization procedure and subsequent fractionation protocol for the preparation of nuclear protein extracts from frozen porcine left ventricular (LV) tissue. This method gave a highly reproducible protein yield (6.5±0.7% of total protein; mean±SE, n=9) and a 6-fold enrichment of the nuclear marker protein B23. The nuclear protein extracts were essentially devoid of cytosolic, myofilament, and histone proteins. Compared to nuclear extracts from fresh LV tissue, some loss of nuclear proteins to the cytosolic fraction was observed. Using this method, we studied the distribution of tyrosinephosphorylated signal transducer and activator of transcription 3 (PY-STAT3) in LV tissue of animals treated with the β-agonist dobutamine. Upon treatment, PY-STAT3 increased 30.2±8.5-fold in total homogenates, but only 6.9±2.1-fold (n=4, P=0.03) in nuclear protein extracts. Of all PY-STAT3 formed, only a minor fraction appeared in the nuclear fraction. This simple and reproducible protocol yielded nuclear protein extracts that were highly enriched in nuclear proteins with almost complete removal of cytosolic and myofilament proteins. This nuclear protein extraction protocol is therefore well-suited for nuclear proteome analysis of frozen heart tissue collected in biobanks.

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Strategies for revealing lower abundance proteins in two-dimensional protein maps

Cited by 61 publications

References 70 publications

Suction blister fluid as potential body fluid for biomarker proteins

Suction blister fluid as potential body fluid for biomarker proteins

Biochemical and proteomic investigation of bovine nasal secretion

Nuclear protein extraction from frozen porcine myocardium

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