Reverse proteomic

Polymer monolithic columns with small diameter have been successfully employed for proteome analysis. Karger and coworkers reported MALDI-TOF of separated fractions spotted on a plate from a polymeric reversed-phase column that showed high peak capacity (Chen et al., 2005). Huber and coworkers reported separation and detection of about 200 peaks within 5 min by using a PSDVB column (Premstaller et al., 2001). 

In an off-line configuration, a complex peptide mixture from a proteomic sample is loaded onto a SCX column and fractions collected (Fig. 11.1). After the collection of fractions, they are then loaded into an autosampler and analyzed via the traditional RP/ MS/MS approach. Using this system, a variety of buffers and elution conditions may be used (Table 11.1). For example, one may use a volatile salt such as ammonium formate (Adkins et al., 2002 Blonder et al., 2004 Fujii et al., 2004 Yu et al., 2004 Qian et al., 2005a and b) or ammonium acetate (Cutillas et al., 2003 Coldham and Woodward, 2004), collect SCX fractions, lyophilize, resuspend in low acetonitrile and acid, and then directly analyze via RP/MS/MS. In most of the cases, when ammonium acetate or ammonium formate are used, a 20-minute wash period is used to remove the ammonium acetate or ammonium formate prior to the reversed-phase gradient (Table 11.1). However, because fractions are collected and can be buffer exchanged,... 

Chen, J., Lee, C.S., Shen, Y., Smith, R.D., Baehrecke, E.H. (2002). Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation. Electrophoresis 23, 3143-3148. 

Davis, M.T., Beierle, J., Bures, E.T., McGinley, M.D., Mort, J., Robinson, J.H., Spahr, C.S., Yu, W., Luethy, R., Patterson, S.D. (2001). Automated LC-LC-MS-MS platform using binary ion-exchange and gradient reversed-phase chromatography for improved proteomic analyses. J. Chromatogr. B Biomed Sci. Appl. 752, 281-291. 

Quadroni M et al. Proteome mapping, mass spectrometric sequencing and reverse transcription-PCR for characterization of the sulfate starvation-induced response in Pseudomonas aeruginosa PAOl. Eur J Biochem 1999 266 986-996. 

In summary, studies carried out with tissue surrogates25 highlight some of the problems that must be overcome before proteins extracted from FFPE tissues can be used for routine proteomic studies. First, these studies demonstrate that reversal of protein-formaldehyde adducts does not assure quantitative extraction of proteins from FFPE tissues or vice-versa. It may ultimately turn out that there is no one universal method that can accomplish both tasks, but that instead, each step will need to be optimized separately. Studies with tissue surrogates also suggest that failure to quantitatively extract the entire protein component from FFPE tissues may result in sampling bias due to the preferential extraction of certain proteins. This behavior may be linked to protein physical properties, such as the isoelectric point. The results of our... 

Wang W, Guo T, Rudnick PA, et al. Membrane proteome analysis of micro-dissected ovarian tumor tissues using capillary isoelectric focusing/reversed-phase liquid chromatography-tandem MS. Anal. Chem. 2007 79 1002-1009. 

Piggott AM, Karuso P. (2008) Rapid identification of a protein binding partner for the marine natural product kahalalide F by using reverse chemical proteomics. ChemBioChem 9 524-530. 

Nishizuka, S., Charboneau, L., Young, L., Major, S., Reinhold, W.C., Waltham, M., Kouros-Mehr, H., Bussey, K.J., Lee, J.K., Espina, V., Munson, RJ., Petricoin, E., Liotta, L.A., and Weinstein, J.N., Proteomic profiling of the NCI-60 cancer cell lines using new high-density reverse-phase lysate microarrays, Proc. Natl. Acad. Sci. USA, 24, 14229-14234, 2003. 

There are several important advantages RPMAs have over antibody arrays and other proteomic techniques such as immunohis-tochemistry or tissue arrays. Antibody arrays usually require a second specific antibody, made in a different species, for each captured protein to be visualized in a manner analogous to enzyme-linked immunosorbent assays (ELISA). Therefore, it becomes difficult to simultaneously optimize the antibody-antigen hybridization conditions for so many antibodies at once present on antibody arrays while minimizing nonspecific cross-reactivity and ensuring that proteins over a wide range of concentrations can be quantitated in a linear fashion (14). Antibody arrays also consume or require much higher inputs of protein than reverse phase arrays. With antibody arrays. 

Brase JC, Mannsperger H, Frohlich H et al (2010) Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification. Proteome Sci 8 36... 

Sheehan KM, Calvert VS, Kay EW et al (2005) Use of reverse phase protein microarrays and reference standard development for molecular network analysis of metastatic ovarian carcinoma. Mol Cell Proteomics 4 346-355... 

Hennessy BT, Lu Y, Gonzalez-Angulo AM et al (2010) A technical assessment of the utility of reverse phase protein arrays for the study of the functional proteome in non-microdis-sected hiunan breast cancers. Clin Proteomics 6 129-151... 

Monolithic silica C,8 reversed phase Chromolith CapRod Merck KgaA 150x0.1 mm Proteome research, analysis of peptides and protein digests... 

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