A novel four- channel multiplexed electrospray liquid chromatography interface is described. This device has been used to analyse both single components and mixtures by liquid chromatography/mass spectrometry (LC/MS) as well as synthetic samples prepared by automated procedures. These data provided unambiguous molecular weight assignments to both major components and synthetic by-products in these samples. In this work particular attention has also been paid to the elimination of interchannel crosstalk. Copyright 1999 John Wiley & Sons, Ltd.

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A study has been undertaken to evaluate the usefulness of MALDI Q-TOF data for protein identification. The comparison of MS data of protein digests obtained on a conventional MALDI TOF instrument to the MS data from the MALDI Q-TOF reveal peptide patterns with similar intensity ratios. However, comparison of MS/MS Q-TOF data produced by nanoelectrospray versus MALDI reveals striking differences. Peptide fragment ions obtained from doubly charged precursors produced by nanoelectrospray are mainly y-type ions with some b-ions in the lower mass range. In contrast, peptide fragment ions produced from the singly charged ions originating from the MALDI source are a mixture of y-, b-and a-ions accompanied by ions resulting from neutral loss of ammonia or water. The ratio and intensity of these fragment ions is found to be strongly sequence dependent for MALDI generated ions. The singly charged peptides generated by MALDI show a preferential cleavage of the C-terminal bond of acidic residues aspartic and glutamic acid and the N-terminal bond of proline. This preferential cleavage can be explained by the mobile proton model and is present in peptides that contain both arginine and an acidic amino acid. The MALDI Q-TOF MS/MS data of 24 out of 26 proteolytic peptides produced by trypsin or Asp-N digestions were successfully used for protein identification via database searching, thus indicating the general usefulness of the data for protein identification. De novo sequencing using a mixture of 16 . This requires only small amounts of protein, provides a relatively fast identification and can easily be automated. In the second approach, fragment ion masses of one or more proteolytic peptides from a protein digest are determined, usually by electrospray ionization tandem mass spectrometry (ESI MS/MS). Database searches can then be performed with different algorithms; either by using partly interpreted data or by submitting the list of fragment ions taken from the MS/MS spectra without any data interpretation [2,3]. However, both approaches suffer from some limitations. In peptide mass fingerprinting, results obtained are not always unambiguous and the method does not cope well with protein mixtures. On the other hand,

2-Aminoacridone (2-AMAC) has been used to derivatize mixtures of N-linked oligosaccharides released from alpha(1)-acid glycoprotein and immunoglobulin G. In each case, the HPLC profile obtained for the derivatized glycans was compared to that obtained after digestion with sialidase and a two-enzyme array system made up of sialidase and alpha-fucosidase, prior to derivatization by 2-AMAC. These studies are rapid and provide a wealth of preliminary information about the degree of sialylation and core fucosylation in the corresponding parent glycans. Moreover, collection of glycans from one single injection has provided enough material for molecular weight determination by MALDI-MS analysis. In this study we have also carried out limited MS-MS studies on enriched fractions of 2-AMAC-glycans using a nanospray orthogonal quadrupole time-of-flight mass spectrometer.

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The separation of trace impurities in cimetidine drug substance by high pressure liquid chromatography (HPLC) showed a series of minor components. These were analysed using an on-line electrospray source connected to a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Working typically with a resolution of 10 000-20 OOO accurate mass measurement of the components confirmed their empirical formulae to generally better than 5ppm accuracy. This work shows that the combination of conventional analytical HPLC using 4.6 mm columns with a low flow rate electrospray source interfaced to an FT-ICR mass spectrometer enables structural information to be obtained on low levels of low mass components.