Effect of the mobile phase composition on the separation and detection of intact proteins by reversed-phase liquid chromatography-electrospray mass spectrometry.

Various buffers (ammonium acetate, ammonium formate, and ammonium hydrogencarbonate), acids (formic acid, acetic acid, heptafluorobutyric acid, and trifluoroacetic acid), and bases (ammonium hydroxide and morpholine) covering the range from 2 to 11.5 have been investigated for their performance in the separation of proteins by reversed-phase liquid chromatography (RPLC) and in their detection by electrospray mass spectrometry (ESI-MS). These additives were first tested for the detection of standard proteins by ESI-MS by flow-injection analysis (FIA). Those additives yielding the highest signals were employed for the separation of standard proteins by using three different reversed-phase columns: two C18 columns (4.6 mm I.D. and 2.1 mm I.D.) and one perfusion column (2 mm I.D.). The sensitivity of the LC-MS system was evaluated with the column giving the best results and with those LC eluents enabling the LC separation of the proteins and also yielding the highest MS signals. For that purpose, calibration curves were compared for both LC-MS and FIA-MS. Formic acid was the additive yielding the highest responses in FIA-MS and trifluoroacetic acid (TFA) gave the best separation and recovery of the proteins. However, problems related to poor recovery of the proteins in the column when formic acid was used and the significant signal suppression observed in MS when TFA was employed, made neither of them suitable for the sensitive detection of the proteins in LC-MS.

Gas-phase ion-molecule reactions of the CH3O (+) cation.

The methoxy cation, CH30(+), formed by collision-induced charge reversal of methoxr anions with a kinetic energy of 8 keY, has been differentiated from the isomenric CH2OH(+) ion by performing low kinetic energy ion-molecule reactions In the radiofrequency-only quadrupole of a reverse-geometry double-focusing quadrupole hybrid mass spectrometer. The methoxy cation reacts with CH3SH, CH3-CH=CH2, (CH3)2O, and CH3CH2Cl by electron transfer, whereas the CH2OH(+) ion reacts by proton transfer with these substrates.

FAB and tandem mass spectrometry for endorphin- and ACTH peptides of molecular weight to 2000.

Four beta-endorphins (beta-endorphin 6-17, 2-17, 1-16, and 1-17) and two adrenocorticotropic hormone (ACTH) peptides (ACTH 1-10 and (1-16)-NH2) were studied by using fast atom bombardment coupled with tandem mass spectrometry. The capability to reproduce metastable ion and collisionally activated decomposition spectra on two different commercial sector mass spectrometers in two different laboratories was found to be acceptable (deviations in relative abundance are less than +/- 50%). The endorphin peptides fragment metastably or upon collisional activation to give abundant B-series ions as well as Y-series ions, whereas Y-series ions are the principal ionic species produced upon the desorption by fast atom bombardment. The ACTH peptides also fragment to give Y-series ions, but of relatively low abundance compared to those from the endorphins. For both sets of peptides, high-energy collisionally activated decomposition and metastable ion decomposition daughter ion spectra are precise, structurally informative--even for peptides up to m/z 2000--and complementary to spectra of daughter ions produced by desorption ionization alone.