Peptide enrichment and protein fractionation using selective electrophoresis

Ly, Linda; Wasinger, Valerie C.

doi:10.1002/pmic.200701088

Cited by 22 publications

(17 citation statements)

References 67 publications

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“…These methods of prefractionation and trapping can be applied to other samples with high-abundance proteins, such as cerebral spinal fluid or urine, to aid in the search for disease biomarkers. This study complements the previous study by Ly and Wasinger [14] in which the MF10 was used to enrich the peptide content of plasma samples prior to analysis by MS. Therefore, the MF10 provides a simple and efficient method for prefractionation of a challenging sample, plasma, under native conditions which is directly compatible with subsequent proteomic analysis and thus providing a further technique to increase the depth of proteome investigation.…”

Section: Proteomics and 2-desupporting

confidence: 52%

New method for prefractionation of plasma for proteomic analysis

Fitzgerald¹,

Walsh²

2010

Electrophoresis

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The depth of proteome analysis is severely limited in complex samples with a wide dynamic range of protein abundance such as plasma. Removal of high-abundance proteins should reveal the signal of lower abundance plasma proteins. However, smaller proteins may be part of larger protein complexes and hence the removal of proteins involved in complexes with high-abundance proteins such as albumin may inhibit the search for disease biomarkers. Prefractionation of a sample divides it into fractions of reduced complexity, allowing improved detection of lower abundance proteins. Using a prefractionation device, which employs Gradiflow™ technology, we were able to separate small volume plasma samples into multiple fractions based on the molecular weight and/or charge. The resulting samples of reduced complexity were directly compatible with 2-DE. The use of this prefractionation machine may therefore be useful in the hunt for disease biomarkers.

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Section: Proteomics and 2-desupporting

confidence: 52%

New method for prefractionation of plasma for proteomic analysis

Fitzgerald¹,

Walsh²

2010

Electrophoresis

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“…At present hundreds to thousands of endogenous peptides, including peptides from low abundance blood proteins, can be routinely extracted and identified from blood fluids (Tucholska et al, 2007;Williams et al, 2007;Hu et al, 2009a). There has been an intense effort to identify (Marshall et al, 2003 and examine the low molecular mass polypeptides of blood, sometimes referred to as the peptidome (Verhaert et al, 2001;Jurgens & Schrader, 2002;Tirumalai et al, 2003;Merrell et al, 2004;Villanueva et al, 2004Villanueva et al, , 2006aVillanueva et al, ,b, 2007Villanueva et al, , 2008Ping et al, 2005;Rai et al, 2005;Tammen et al, 2005aTammen et al, ,b, 2007Falth et al, 2006Falth et al, , 2008Geho et al, 2006;Govorukhina et al, 2006;Hortin, 2006;Ogata et al, 2006;Huang et al, 2006b;Zheng, Baker, & Hancock, 2006b;Aristoteli, Molloy, & Baker, 2007;Tian et al, 2007;Tomonaga & Nomura, 2007;Tucholska et al, 2007;Hernandez et al, 2008;Kay et al, 2008;Kojima et al, 2008;Ly & Wasinger, 2008;Mayer, Poirier, & Seidah, 2008;Navare et al, 2008;Pernemalm et al, 2008;Plavina et al, 2008;Ward et al, 2008;Xia, Wu, & Jemal, 200...…”

Section: Mass Spectromtery Of Blood Endogenous Polypeptidesmentioning

confidence: 96%

“…Endogenous peptides have been fractionated by size and pH using selective electrophoresis (Ly & Wasinger, 2008). Identified peptides might be confirmed by observing the cleavage patterns of the parent proteins that have been verified by using SDS-PAGE and Western blot (Mansfield, 1995;Marshall et al, 2003).…”

Section: Electrophoresismentioning

confidence: 99%

Mass spectrometry of peptides and proteins from human blood

Zhu

Bowden

Zhang

et al. 2010

Mass Spectrometry Reviews

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It is difficult to convey the accelerating rate and growing importance of mass spectrometry applications to human blood proteins and peptides. Mass spectrometry can rapidly detect and identify the ionizable peptides from the proteins in a simple mixture and reveal many of their post-translational modifications. However, blood is a complex mixture that may contain many proteins first expressed in cells and tissues. The complete analysis of blood proteins is a daunting task that will rely on a wide range of disciplines from physics, chemistry, biochemistry, genetics, electromagnetic instrumentation, mathematics and computation. Therefore the comprehensive discovery and analysis of blood proteins will rank among the great technical challenges and require the cumulative sum of many of mankind's scientific achievements together. A variety of methods have been used to fractionate, analyze and identify proteins from blood, each yielding a small piece of the whole and throwing the great size of the task into sharp relief. The approaches attempted to date clearly indicate that enumerating the proteins and peptides of blood can be accomplished. There is no doubt that the mass spectrometry of blood will be crucial to the discovery and analysis of proteins, enzyme activities, and post-translational processes that underlay the mechanisms of disease. At present both discovery and quantification of proteins from blood are commonly reaching sensitivities of ∼1 ng/mL.

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“…Fifteen microliters of (10%) protease inhibitor (Roche, Basel, Switzerland) was added to 150 l of the mixed pools of plasma resulting in 10 pools of grouped samples comprising of (22). Here, only the 1 to 25 kDa fraction is further reported.…”

Section: Methodsmentioning

confidence: 99%

“…Such partitioning of proteins enabled powerful enrichment of low mass and poorly abundant proteins (22). Using a shotgun proteomic approach, this large scale survey of proteins has highlighted the increase in inflammatory and acute phase proteins that are known to plague the illness and in addition has revealed novel peptides and proteins that can be used to discriminate IBD from controls, and UC from CD.…”

Section: Inflammatory Bowel Disease (Ibd)mentioning

confidence: 99%

Low Mass Blood Peptides Discriminative of Inflammatory Bowel Disease (IBD) Severity: A Quantitative Proteomic Perspective

Wasinger

Yau

Duo

et al. 2016

Molecular & Cellular Proteomics

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Breakdown of the protective gut barrier releases effector molecules and degradation products into the blood stream making serum and plasma ideal as a diagnostic medium. The enriched low mass proteome is unexplored as a source of differentiators for diagnosing and monitoring inflammatory bowel disease (IBD) activity, that is less invasive than colonoscopy. Differences in the enriched low mass plasma proteome (<25 kDa) were assessed by label-free quantitative mass-spectrometry. A panel of marker candidates were progressed to validation phase and "Tier-2" FDA-level validated quantitative assay. Proteins important in maintaining gut barrier function and homeostasis at the epithelial interface have been quantitated by multiple reaction monitoring in plasma and serum including both inflammatory; rheumatoid arthritis controls, and non-inflammatory healthy controls; ulcerative colitis (UC), and Crohn's disease (CD) patients. Detection by immunoblot confirmed presence at the protein level in plasma. Correlation analysis and receiver operator characteristics were used to report the sensitivity and specificity. Peptides differentiating controls from IBD originate from secreted phosphoprotein 24 (SPP24, p ‫؍‬ 0.000086, 0.009); whereas those in remission and healthy can be differentiated in UC by SPP24 (p ‫؍‬ 0.00023, 0.001), ␣-1-microglobulin (AMBP, p ‫؍‬ 0.006) and CD by SPP24 (p ‫؍‬ 0.019, 0.05). UC and CD can be differentiated by Guanylin (GUC2A, p ‫؍‬ 0.001), and Secretogranin-1 (CHGB p ‫؍‬ 0.035). Active and quiescent disease can also be differentiated in UC and CD by CHGB (p < 0.023) SPP24 (p < 0.023) and AMBP (UC p ‫؍‬ 0.046). Five peptides discriminating IBD activity and severity had very little-to-no correlation to erythrocyte sedimentation rate, C-reactive protein, white cell or platelet counts. Three of these peptides were found to be binding partners to SPP24 protein alongside other known matrix proteins. These proteins have the potential to improve diagnosis and evaluate IBD activity, reducing the need for more invasive techniques. Data are available via ProteomeXchange with identifier

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Peptide enrichment and protein fractionation using selective electrophoresis

Cited by 22 publications

References 67 publications

New method for prefractionation of plasma for proteomic analysis

New method for prefractionation of plasma for proteomic analysis

Mass spectrometry of peptides and proteins from human blood

Low Mass Blood Peptides Discriminative of Inflammatory Bowel Disease (IBD) Severity: A Quantitative Proteomic Perspective

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