Reverse phase liquid chromatography Proteomics

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. 

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. 

Shen, Y. R, Zhao, R., Belov, M. E., Conrads, T. R, Anderson, G. A., Tang, K. Q., Pasa-Tolic, L., Veenstra, T. D., Lipton, M. S., Udseth, H. R., and Smith, R. D., Packed capillary reversed-phase liquid chromatography with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for proteomics. Analytical Chemistry 73(8), 1766-1775, 2001. 

Figure 4 Depiction of shot-gun proteomics using multidimensional protein identification technology a complex mixture of peptide fragments in the digest are resolved by a combination of ion-exchange and reversed-phase liquid chromatography.

Shen, Y. Zhao, R. Belov, M.E. Conrads, T.P. Anderson, G.A. Tang, K. Pasa-Tolic, L. Veenstra, T.D. Lipton, M.S. Udseth, H.R. Smith, R.D. Packed Capillary Reverse-Phase Liquid Chromatography with High-Performance Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Proteomics, Anal. Chem. 12>, Yl(>C-Vn5 (2001). 

Lam MPY, Law CH, Quan Q, et al. Fully automatable multidimensional reversed-phase liquid chromatography with online tandem mass spectrometry. In Marlins-de-Souza D editor. Shotgun proteomics methods and protocols, methods in molecular biology. Vol. 1156. New York Springer 2014. p. 39-51. doi 10.1007/978-l-4939-0685-7 3. 

Lee, Y. H., Kim, M. S., Choie, W. S., Min, H. K., Lee, S. W. (2004) Hi y informative proteome analysis by combining improved N-terminal sulfonation for de novo peptide sequencing and online capillary reverse-phase liquid chromatography/tandem mass spectrometry. Proteomics, 4, 1684-1694. 

The peptide mixture that results from tryptic digestion of a protein mixmre is usually fractionated prior to being submitted to mass spectrometric analysis. The goal of the separation is to reduce the complexity of the peptide mixmre to simplify analysis of peptide mass peaks. The most commonly used method in exosome proteome studies is reverse phase high performance liquid chromatography (Feviier et al. 2004 Pisitkun et al. 2004 Potolicchio et al. 2005 Segura et al. 2005 Faure et al. 2006). For this method, a mixture of peptides in aqueous solution is loaded onto a colurrm packed with... 

The methods for each study are divided into the initial protein separation step, a second separation step if applicable, the type of mass analysis, and the software used for peptide identification. ID = one dimensional polyacrylamide gel electrophoresis, 2D = two dimensional polyacrylamide gel electrophoresis, MS = mass spectrometry (peptide mass fingerprinting), MS/MS = tandem mass spectrometry, MALDI-TOF = matrix assisted laser desorption/ionization-time of flight, MS FIT = software from Protein Prospector, http //prospector.ucsf edu/, ESI = electrospray ionization, Q-TOF = quadrupole-time of flight, PPSS2 =Protana s Proteomic Software Suite (ProtanaEngineering, Odense, Denmark), Mascot = Matiix Science, http //www.matrixscience.com/, TOF-TOF = MALDI plus TOF tandem mass spectrometry, RP-HPLC = reverse phase high performance liquid chromatography, Q-IT = quadrupole ion trap, LIT = linear ion trap. Bioworks = Thermo Electron Corporation. 

Sheng S, Chen D, van Eyk JE (2006) Multidimensional liquid chromatography separadon of intact proteins by chromatographic focusing and reversed phase of the human serum proteome Optimizadon and protein database. Mol Cell Proteomics 5 26-34. 

Although liquid chromatography techniques have become quite popular in the separation of peptides in complex protein digests, they are yet to make an impact for the separation of protein samples for proteome-wide applications. It is envisioned that in the future their application for protein separation will increase. Various combinations of reversed-phase (RP)-HPLC with ion-exchange, size-exclusion, chromato-focusing (CF), IEF, and capillary electrophoresis (CE) have emerged for 2D separation of complex mixtures of proteins and peptides. A recent addition in this field is the use of CF as the first dimension and RP-FIPLC as the second-dimension separation device.14 CF is a column-based liquid-phase separation technique, in which proteins are fractionated on the basis of differences in their p/values in a weak ion-exchange column. 

Proteomics, protein abundance, function, and interactions within the cell, can be measured by liquid chromatography (6) and mass spectrometry or by reverse-phase protein arrays. The interactions are done by yeast-two-hybrid, cochromatography, and other methods. 

Badock V, Steinhusen U, Bommert K, et al. (2001). Prefractionation of protein samples for proteome analysis using reversed-phase high-performance liquid chromatography. Electrophor. 22 2856-2864. 

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