Proteins reverse phase chromatograph

Lau, S. Y. M., Taneja, A. K., and Hodges, R. S., Effects of high-performance liquid chromatographic solvents and hydrophobic matrices on the secondary and quaternary structure of a model protein. Reversed-phase and size exclusion high-performance liquid chromatography, /. Chromatogr., 317, 129, 1984. 

Cohen, K. A., Schellenberg, K., Benedek, K., Karger, B. L., Grego, B., and Hearn, M. T. W., Mobile-phase and temperature effects in the reversed phase chromatographic separation of proteins, Anal. Biochem., 140, 223, 1984. 

Various applications of PITC in analyses of acid hydrolysates of purified proteins and peptides (91) and of hydrolysates of feeds (92) have been described. Bidlingmeyer et al. (93) showed reversed-phase chromatographic analysis of free amino acids and hydrolysates of foods (soybean flour, mozzarella cheese, beer, and soy sauce) with formation of PITC derivatives to be a fast, reproducible method that presented a very good correlation with the results obtained by ion-exchange chromatography. 

At about the same time, our laboratory has reported the development and validation of an LC tandem MS assay for as much as six TKIs simultaneously. The proposed LC-MS/MS method allows the simultaneous determination of clinically relevant ranges of concentrations for the six major TKIs currently in use imatinib, dasatinib, nilotinib, sunitinib, sorafenib, and lapatinib [122], Plasma is purified by acetonitrile protein precipitation followed by reversed-phase chromatographic separation. Analyte quantification is performed by electrospray ionization-triple quadrupole mass spectrometry by selected reaction monitoring (SRM) detection using the positive mode. This was the first broad-range LC-MS/MS assay covering the major currently in-use TKIs. 

Here again, not all chromatographic setups are usable for any proteomics question. The use of protein reverse phase chromatography, which has been advocated for plasma proteomics (Moritz et al. 2005), precludes in turn the use of any detergent of any type. This prevents the use of this chromatographic setup in most subcellular proteomics experiments, where detergents must be used to solubilize the membrane limiting the subcellular compartments. 

Figure 1. Reverse-phase chromatographic maps of tryptic digests of reduced caiboxymethylated ribonuclease preparations (a) RNase A control (b) RNase S control and (c) RNase S treated for 1 min with C2N2. Modified and unmodified protein samples were analyzed by reverse-phase HPLC (Perkin-Elmer 250 Binary Pumping Model 235, Diode array Detector, Vydac C-18, 18TP54 Column) using the method of McWherter et al. (18). The sample digests were frozen and stored at -70°C until analyzed.

D. Reverse phase chromatographic separation of penicillin binding proteins (PBPs) and cross-linked PBP-morphogene products (See Scheme 2)... 

Fluorescamine (Fluram). Fluorescamine was developed from the study of ninhydrin chemistry as a reagent for detecting amino groups (Udenfriend et al., 1972) and has been successfully used in the reversed phase chromatographic analysis of proteins and peptides using an on-line post-column detection system. This reagent requires two separate pumps for the delivery of the mobile phase buffer and the reagent and this method may therefore be considered to be less... 

Reversed-phase chromatographic separatiorr irwolves loading of a protein mixture onto a hydrophobic stationary phase in water or a water—solvent mixture, usually containing very dilute acid (usually... 

An LC-MS-MS method to monitor lonafarnib (a novel anticancer drug that inhibits farnesyl transferase) in human plasma. Deuterated internal standard is used proteins are precipitated by acetonitrile. Reverse-phase chromatographic separation is performed using acetonitrile/water/formic acid (50 50 0.05, v/v/v) mobile phase. Time of analysis 8 min. A triple quadrupole tandem mass spectrometer in the positive-ion mode with multiple reaction monitoring is used for detection. The cahbration curve has been established in the 2.5-2500 ng/ml concentration range. The validated method was successfully used in phase I trials of the drug. 

AMlc, Dihydro AMI, AM 19, and AM4N) tend to bind to lipoproteins, protein precipitation and solid-phase extraction are necessary prior to reverse-phase chromatographic analysis. The drug and its metabohtes (which exhibit patient-specific patterns) are detected by MS in the form of sodium adducts after EL Hepatotoxic potential has been confirmed and strong correlation between AM19 and CRP and IL6 observed. 

Reversed-phase high performance Hquid chromatography has come into use for estimating the purity of proteins and peptides as weU. However, before employed, a high performance Hquid chromatographic (hplc) profile of a given protein must be completely vaHdated (43). 

Despite the intrinsically nonspecific nature of ion-exchange and reversed-phase/hydrophobic interactions, it is often found that chromatographic techniques based on these interactions can exhibit remarkable resolution this is attributed to the dynamics of multisite interactions being different for proteins having differing surface distributions of hydrophobic and/or ionizable groups. 

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