Unseen Proteome:  Mining Below the Tip of the Iceberg To Find Low Abundance and Membrane Proteins

Pedersen, Susanne K.; Harry, Jenny L.; Sebastian, Lucille; Baker, Jasmine; Traini, Mathew; McCarthy, John; Manoharan, Abi; Wilkins, Marc R.; Gooley, Andrew A.; Righetti, Pier Giorgio; Packer, Nicolle H.; Williams, Keith L.; Herbert, Ben R.

doi:10.1021/pr025588i

Cited by 136 publications

(106 citation statements)

References 39 publications

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“…Localization of identified proteins was determined by a combination of peer-reviewed literature search, NCBI protein database search [39], PSORT subcellular localization prediction [40] and prediction of trans-membrane protein segments using HMMTOP [39][40][41] (Tables 1 and 2). …”

Section: Image Analysis and Protein Identificationmentioning

confidence: 99%

Immunoproteomic identification of bovine pericardium xenoantigens

et al. 2008

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Bovine pericardium is an important biomaterial with current application in glutaraldehyde-fixed bioprosthetic heart valves and possible future application as an unfixed biological scaffold for tissue engineering. The importance of both humoral and cell-mediated rejection responses toward fixed and unfixed xenogeneic tissues has become increasingly apparent. However, the full scope and specific identities of bovine pericardium proteins that can elicit an immune response remain largely unknown. In this study, an immunoproteomic approach was used to survey bovine pericardium proteins for their ability to elicit a humoral immune response in rabbits. A two-stage protein extraction protocol was used to separate bovine pericardium proteins into water-and lipid-soluble fractions. Two-dimensional gel electrophoresis was performed to separate the proteins from each fraction. Western blots were generated from two-dimensional gels of both bovine pericardium protein fractions. These blots were probed with serum from rabbits immunized with bovine pericardium and a secondary antibody was used to assess for IgG positivity. Western blots were compared to duplicate two-dimensional gels and proteins in matched spots were identified by tandem mass spectrometry. Thirty-one putative protein antigens were identified, eight of which are known to be antigenic from previous studies. All of the putative antigens demonstrated progressive staining intensity with increasing days of post-exposure serum. Identified antigenic proteins represented a variety of functional and structural protein types, and included both cellular and matrix proteins. The results of this study have implications for the use of bovine pericardium as a biomaterial in bioprostheses and tissue engineering applications, as well as xenotransplantation in general.

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Section: Image Analysis and Protein Identificationmentioning

confidence: 99%

Immunoproteomic identification of bovine pericardium xenoantigens

et al. 2008

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“…It should be noted that a whole family of nondetergent solubilizers, including glycols, sugars, and common zwitterions such as taurine, has been reported to greatly improve protein solubility in the proximity of their pIs [54][55][56], particularly, for the analysis of membrane proteins using 2-D PAGE. These additives may be employed for completely preventing or largely alleviating the solubility issue, which may be encountered during the CIEF separation.…”

Section: Cief-based Multidimensional Separationsmentioning

confidence: 99%

Capillary separations enabling tissue proteomics‐based biomarker discovery

et al. 2006

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Development of the capability to enable large‐scale proteome studies, analogous to comprehensive gene expression analysis, will clearly have far‐reaching impacts on protein biomarker investigations of human diseases such as cancer through interrogation of the archived fresh frozen and formalin‐fixed and paraffin‐embedded tissue collections. This review therefore focuses on the most recent advances in microdissection techniques and proteome platforms for procuring homogeneous subpopulations of tumor cells or structures and performing comprehensive analysis of protein profiles within tissue specimens, respectively. Developments in capillary separations capable of providing extremely high resolving power and selective analyte enrichment are particularly highlighted for their roles within the broader context of a state‐of‐the‐art integrated tissue proteome effort. The capabilities of CIEF‐based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. These proteome technological advances combined with recently developed tissue microdissection techniques provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.

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“…Pedersen applied the same technology to fractionate alkaline proteins from Saccharomyes cerevisiae solubilized-membrane protein mixtures within the MCE compartment between pH 7.5 and 10.5. The concentrated alkaline fraction was subjected to narrow-pH range 2D PAGE followed by MALDI-TOF MS (Pedersen et al, 2003). A total of 93 unique proteins were identified in this pH 7-10.5 fraction, including 30 low-abundance proteins with the codon adaptation index (CAI) below 0.2, 20 integral membrane proteins, and 10 membrane-associated proteins.…”

Section: A Two Dimensional Gel-based Separationsmentioning

confidence: 99%

Intact‐protein based sample preparation strategies for proteome analysis in combination with mass spectrometry

Wang

Hanash

2004

Mass Spectrometry Reviews

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The complexity of tissue and cell proteomes and the vast dynamic range of protein abundance present a formidable challenge for analysis that no one analytical technique can overcome. As a result, there is a need to integrate technologies to achieve the high-resolution and high-sensitivity analysis of complex biological samples. The combined technologies of separation science and biological mass spectrometry (Bio-MS) are the current workhorse in proteomics, and are continuing to evolve to meet the needs for high sensitivity and high throughput. They are relied upon for protein quantification, identification, and analysis of post-translational modifications (PTMs). The standard technique of two dimensional poly-acrylamide gel electrophoresis (2D PAGE) offers relatively limited resolution and sensitivity for the simultaneous analysis of all cellular proteins, with only the most highly abundant proteins detectable in whole cell or tissuederived samples. Hence, many alternative strategies are being explored. Numerous sample preparation procedures are currently available to reduce sample complexity and to increase the detectability of low-abundance proteins. Maintaining proteins intact during sample preparation has important advantages compared with strategies that digest proteins at an early step. These strategies include the ability to quantitate and recover proteins, and the assessment of PTMs. A review of current intact protein-based strategies for protein sample preparation prior to mass spectrometry (MS) is presented in the context of biomedically driven applications. # 2004 Wiley Periodicals, Inc., Mass Spec Rev 24: [413][414][415][416][417][418][419][420][421][422][423][424][425][426] 2005

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Unseen Proteome: Mining Below the Tip of the Iceberg To Find Low Abundance and Membrane Proteins

Cited by 136 publications

References 39 publications

Immunoproteomic identification of bovine pericardium xenoantigens

Immunoproteomic identification of bovine pericardium xenoantigens

Capillary separations enabling tissue proteomics‐based biomarker discovery

Intact‐protein based sample preparation strategies for proteome analysis in combination with mass spectrometry

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