HPLC/MALDI-TOF

Se-enriched yeast Water extraction followed by tryptic digestion SEC, reversed-phase HPLC, MALDI-TOF-MS, ESI-MS, ESI-MS-MS... 

Lasaosa M, Delmotte N, Huber CG, et al. A 2D reversed-phase x ion-pair reversed-phase HPLC-MALDI TOF/TOF-MS approach for shotgun proteome analysis. Anal Bioanal Chem. 2009 393 1245-56. doi 10.1007/s00216-008-2539-1. 

Wall, D. B. Kachman, M. T. Gong, S. Hinderer, R. Paras, S. Misek, D. E. Hanash, S. M. Lubman, D. M. Isoelectric focusing nonporous RP HPLC A two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry. Anal. Chem. 2000, 72, 1099-1111. 

Proteomics ultimately hinges upon protein identification to reveal the meaning behind the masses, spots, or peaks detected by other means. Because fraction collection is a natural component of HPLC separations, intact proteins can be readily collected either for direct analysis or for proteolytic digestion and identification using peptide mass fingerprinting (PMF) in conjunction with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). 

GPC/SEC, MALDI-MS, membrane osmometry, vapour pressure osmometry, viscometry, light scattering, TDFRS, SAXS, SANS, SEC-HPLC, SEC-MS, SEC-IR, FFF, ultracentrifugation, MALDI-TOF-MS, NMR, capillary electrophoresis... 

Liquid chromatography (LC) encompasses several techniques, which includes HPLC and SEC (sometimes referred to as GPC). Many variants of and developments from these techniques exist, and are used in the study and analysis of polymer degradation/oxidation. As discussed earlier, SEC is often coupled with MALDI-ToF-MS to facilitate the identification of the products of polymer degradation. SEC has also been coupled with mid-infrared detection and similarly used for studies of polymer degradation. SEC/GPC is discussed further below. 

Matrix-associated laser desorption ionization with a time-of-flight mass analyser (MALDI-ToF) was used to examine the crude tryptic peptide mixture from a number of the proteins, without HPLC separation, to provide a mass map, i.e. a survey of the molecular weights of the peptides generated by the digestion process. 

The 4 mM soln of the crude peptide [H-(Gly-Pro-Hyp)5-Gly-Pro-Gln-Gly-Leu-Leu-Gly-Ala-Hyp-Gly-Ile-Leu-Gly-Cys(Acm)-Cys-Gly-Gly-OH] 28 in degassed argon-sat. DMF/AcOH (95 5) was added dropwise to a 100 mM soln of di[5-nitro(2-pyridyl)]disulfide (5 equiv) in DMF/AcOH (95 5) with exclusion of air oxygen. The reaction was monitored spectroscopically at 430 nm, and after completion (1 to 2 h), the solvent was removed under reduced pressure. The resulting residue was dissolved in H20 and the excess reagent was filtered off. The H20 was removed under reduced pressure and the residue was reprecipitated from TFE with methyl /ert-butyl ether and purified by preparative HPLC to give 29 yield 13% the product was characterized by MALDI-TOF-MS, HPLC, and amino acid analysis. 

To a 50 mM soln of heterodimer 31 in TFA/AcOH (1 2) freshly prepared Npys-Cl (1.1 equiv) was added. After 30 min the bulk of the solvent was removed under reduced pressure and the residue was purified by gel chromatography to give 32 yield 77% the product was characterized by HPLC and MALDI-TOF-MS. 

The reduced form of Na+, K+-ATPase inhibitor-I (10) was obtained by treatment of the protected peptide synthesized by the soln procedure with HF, followed by reaction with Hg(OAc)2. After purification of the crude product on Sephadex G-25, the reduced peptide (110 mg) was dissolved in 0.1 M NH4OAc buffer (1L, pH 7.8) at a peptide concentration of 0.018 mM and then stirred at rt. After 24 h, the major peak in the HPLC, which coeluted with the natural product, corresponded to 55% of the product distribution. The mixture was acidified to pH 3 with AcOH and 10 was purified by RP-HPLC. When the oxidation was carried out in the presence redox reagents at a peptide/GSH/GSSG ratio of 1 100 10, after 24 h the major oxidation product increased to 69%. The mixture was acidified with AcOH and the product (10) isolated by preparative HPLC yield 20%. The product was characterized by MALDI-TOF-MS and amino acid analysis a combination of enzymatic peptide mapping and synthetic approaches were applied to assign the cystine connectivities. 

Presently, FAB-MS spectra are routinely used to characterize synthetic tyrosine O-sulfate peptides.152,57,63-671 Since partial hydrolysis of the sulfate ester occurs in the gas phase, quantification of the tyrosine O-sulfate residue by mass spectrometry is not possible, but combined with one-peak assignment in HPLC, FAB-MS represents a powerful analytical tool. On the other hand, partial hydrolysis in the gas phase excludes the presence of sul-fonated species which should be perfectly stable. In early studies the presence of such species were excluded by quantitative recovery of tyrosine upon acid hydrolysis or upon hydrolysis with arylsulfatase.1361 Recently, even MALDI-TOF-MS spectra of CCK-peptides1441 and of conotoxins a-PnIA and a-PnlB 138 were reported which show that in the positive-ion mode the [M + H-S03]+ ions represent the base peaks, while in the negative-ion mode, [M-H]-ions consistently correspond to the base peaks. In the CCK peptides intramolecular salt bridging of the sulfate hemi-ester with proximal positive charges of arginine or lysine side chains was found to reduce the extent of hydrolysis in the gas phase significantly.144,1491... 

MALDI-TOF-MS has been used to identify and quantify other anthocyanins in foods.When the anthocyanin content of highbush blueberries at different stages of anthocyanin formation were analyzed by both HPLC and MALDI-TOF-MS, it was found that both techniques provided comparable quantitative anthocyanin profiles. While HPLC could distinguish anthocyanin isomers, MALDI-TOF-MS proved to be more rapid. MALDI-TOF-MS has also been used to identify the isoflavones in soy samples. In a comparison of several matrices, 2, 4, 6 -trihydroxyacetophenone (THAP) and 2,5-dihydroxybenzoic acid... 

Wang, J., Kalt, W., and Sporns, P., Comparison between HPLC and MALDI-TOF MS analysis of anthocyanins in highbush blueberries, J. Agric. Food Chem., 48, 3330, 2000. 

Below we report methodological studies based upon HPLC, GC/FID, GC-MS, LC-MS, matrix-assisted laser desorption ionisation coupled with time-of-flight mass spectrometry (MALDI-ToF/MS), CE, proton nuclear magnetic resonance ( I INMR), RIA and enzymatic colorimetric techniques. 

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