Approaches to functional genomics: potential of matrix-assisted laser desorption ionization--time of flight mass spectrometry combined with separation methods for the analysis of DNA in biological samples.

MALDI-TOF MS has potential as a valuable technique in DNA mapping studies and may well be complementary to other approaches to DNA analysis such as gel electrophoresis and sequencing. This study used 2,6-dihydroxyacetophenone (DHAP) mixed with diammonium hydrogen citrate (DAHC) as the matrix. In addition, recent technical advances such as time lag focussing (TLF) and better selection of matrices (such as 3-hydroxypicolinic acid (3 HPA) and picolinic acid (PA)) extended the range of DNA fragments that can be studied by this approach. The following samples were investigated: Poly-T mixture (dT 15, 19, 20, 25, 74 and 75), plasmid pBR322 derived oligonucleotides (10, 11, 12, 13, 14, 15, 19, 20 and 50 nucleotides long) and DNA fragments of 25, 36 and 37 base pairs corresponding to a fragment in the restriction map for the gene corresponding to the hexon protein of Adenovirus 2 and 5. The results were contrasted with similar analyses performed by ion-paired reversed-phase HPLC coupled to on-line electrospray mass spectrometry.

Reversed-phase high-performance liquid chromatographic assay for the adenovirus type 5 proteome.

An RP-HPLC assay was developed for a recombinant adenovirus type 5. During chromatography, intact adenovirus dissociated into its structural components (DNA and proteins) and the viral proteome was separated yielding a characteristic fingerprint. The individual components were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, N-terminal sequencing and amino acid composition. The assay was utilized to measure adenovirus particle concentration through quantification of structural proteins. Each structural protein provided independent measurement of virus concentration allowing verification of accuracy. The assay sensitivity is at or below 2 x 10(8) particles. Contrary to the benchmark spectrophotometric assay, the RP-HPLC assay was shown to be insensitive to contaminants common for partially purified adenovirus preparations.

Investigation of the potential of capillary electrophoresis with off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for clinical analysis: examination of a glycoprotein factor associated with cancer cachexia.

The potential of capillary electrophoresis (CE) with offline matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has been demonstrated for the examination of a glycoprotein factor associated with cancer cachexia. A comparison of CE profiles of a healthy volunteer and a cancer patient shows the presence of additional peaks in the electropherogram of the cancer patient that could be associated with cachexia. Micropreparative CE was performed with 180-micron fused silica capillary columns with tapered ends to collect CE fractions for further identification by MALDI-TOF-MS. The analysis of crude urine samples of cancer patients exhibiting cachexia, as well as CE fractions, with MALDI-TOF-MS using ferulic acid as the matrix shows a number of characteristic ions at m/z values of approximately 24 and approximately 67 kDa. The 24-kDa peak may be identified as the cachectic factor, a glycoprotein, whereas the peak at 67 kDa is identified as albumin, which is present in urine of most patients, and to which the cachectic factor is noncovalently bound. The combined use of CE and MALDI-TOF-MS was successful in detecting cachexia in all of the patients in this study, including one patient that was in an early phase of the disease.

Application of new analytical technology to the production of a "well-characterized biological".

The role of new analytical technology in the development of the concept of a "Well Characterized Biological" is to provide suitable methodology that allows the characterization of even the most complex protein sample so that a consistent manufacturing process can be established. Glycoproteins are among the most challenging of products to characterize because of extreme sample microheterogeneity due to the carbohydrate moieties. As an example of the appropriate use of new analytical technology this review will examine the steps necessary to demonstrate that a glycoprotein is a "Well Characterized Biological". The key to characterization of complex protein samples lies in the use of appropriate combinations of the different methods that analyse the sample from substantially orthogonal and independent directions. An important advantage of capillary electrophoresis (HPCE) in this application is the complementarily of the technique with reversed phase HPLC (RPLC). Thus mixtures of variants of a polypeptide that are difficult to separate by RPLC can often be readily resolved by HPCE. Both separation techniques are well suited to the analysis of peptide maps, although RPLC is particularly powerful when used in combination with on-line electrospray mass spectrometry (ESI-MS) which allows for the effective ionization and detection of even high MW glycopeptides. In this sense the ESI-MS is an ideal detector for on-line mass detection after a RPLC separation of medium MW fragments (300 to 6000 emu) that are typically present in an enzyme digest of a protein. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOFMS) is a valuable technique for off-line characterization of CE fractions due to the high sensitivity of the method and its tolerance of samples with moderate levels of salt. The development of an effective protocol for the analysis of glycoform populations of intact glycoproteins by a combination of HPCE and off-line MALDI-TOFMS wil be demonstrated by the successful analysis of two highly heterogeneous glycoproteins, ovalbumin and Desmodus Salivary Plasminogen Activator (DSPA).

The use of multidimensional liquid-phase separations and mass spectrometry for the detailed characterization of posttranslational modifications in glycoproteins.

The goal of characterization of the proteome, while challenging in itself, is further complicated by the microheterogeneity introduced by posttranslational modifications such as glycosylation. A combination of liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry (MS) offers the advantages of unique selectivity and high efficiency of the separation methods combined with the mass specificity and sensitivity of MS. In the current work, the combination of liquid-phase separations and mass spectrometry is demonstrated through the on-line coupling of electrospray ionization mass spectrometry (ESI-MS) and off-line coupling with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). LC/ESI-MS yields real-time results while maintaining the separation obtained from the LC analysis. CE/MALDI TOF-MS offers high-mass detection and extremely low detection limits. The unique separation selectivity of CE relative to reversed-phase HPLC separations of the members of a glycopeptide family was used to develop an integrated multidimensional analysis achieved by the off-line coupling of LC, CE, and MALDI TOF-MS. To demonstrate the applicability of these techniques to the characterization of the heterogeneity of posttranslational modifications present in glycoproteins, we will report on the study of the glycoforms present in a N-linked site in a single-chain plasminogen activator (DSPAα1).

Analysis of recombinant DNA-derived glycoproteins via high-performance capillary electrophoresis coupled with off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

This paper describes the analysis of glycoform populations of the glycoproteins ovalbumin and Desmodus salivary plasminogen activator (DSPA alpha 1) by a combination of capillary electrophoresis (CE) and off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Ovalbumin has a single N-linked glycosylation site and DSPA alpha 1 has six sites for potential glycosylation, 2 N-linked and four O-linked. The conditions used for the electrophoretic separation of ovalbumin include a borate buffer system, together with a diamine additive such as 1,4-diaminobutane (DAB). An electropherogram of DSPA glycoforms could be obtained at pH 3.0 (phosphate buffer) using a bovine serum albumin (BSA) coated capillary. Fraction collection was performed by controlled application of pressure [5000 Pa (50 mbar)] for zone elution and MALDI-TOF-MS was performed on samples prepared by a 1:1 dilution with the UV absorbing matrix sinapinic acid. Both electrophoretic separations were successfully characterized by good quality mass spectra and distinct mass trends were observed for the collected fractions. It is likely that each of the collected fractions are still mixtures of glycoforms and explanation of relative mobilities or masses of different fractions is not possible at this stage. The ability to perform rapid off-line MALDI-TOF-MS of fractions from complex electropherograms will be a powerful tool to demonstrate product consistency in the manufacture of glycoprotein pharmaceuticals.

Analysis of Oligonucleotides by HPLC-Electrospray Ionization Mass Spectrometry.

A new interface procedure has been developed that allows, for the first time, the high-efficiency analysis of synthetic oligonucleotides up to 75 bases by reversed-phase HPLC and on-line electrospray ionization mass spectrometry. For oligonucleotides up to 30 bases in length, single-base resolution can be obtained with low levels of cation adduct formation in the negative ion electrospray mass spectra. A key part of the method uses 1,1,1,3,3,3-hexafluoro-2-propanol as an additive to the HPLC mobile phase, adjusted to pH 7.0 with triethylamine. This novel additive results in both good HPLC separation and efficient electrospray ionization. The broad potential of this new method is demonstrated for synthetic homopolymers of thymidine (PolyT), fragments based on the pBR322 plasmid sequence, and phosphorothioate ester antisense oligonucleotides. This approach will be of particular utility for the characterization of DNA probes and PCR primers and quality control of antisense compounds such as phosphorothioates and their metabolites, as well as of materials used in clinical trials.

Application of multidimensional affinity high-performance liquid chromatography and electrospray ionization liquid chromatography-mass spectrometry to the characterization of glycosylation in single-chain plasminogen activator. Initial results.

Preliminary results are presented using a combination of affinity chromatography, reversed-phase HPLC and electrospray ionization mass spectrometry to produced peptide maps for N-linked, O-linked and non-glycosylated peptides from an endoproteinase LysC digest of DSPA alpha 1, a recombinant DNA derived glycoprotein. Although the system was used to identify a number of major N-linked structures, notably complex biantennary structures attached to asparagine 362, no O-linked glycopeptides from the possible 4 attachment sites were identified. The system did, however, demonstrate the feasibility of the approach and the applicability of the instrumental system.

Application of high-performance liquid chromatograph-electrospray ionization mass spectrometry and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry in combination with selective enzymatic modifications in the characterization of glycosylation patterns in single-chain plasminogen activator.

The application of high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective enzymatic deglycosylation treatments is demonstrated in the analysis of glycosylation patterns in recombinant Desmodus salivary plasminogen activator, a heterogeneous glycoprotein. The sample was initially digested with a proteolytic enzyme (endoproteinase Lys-C) and then further treated with either PNGase F to remove N-linked carbohydrates or a combination of neuraminidase and O-glycosidase to remove sialic acid and O-linked carbohydrates. By comparison of the LC-ESI-MS peptide maps for the fully glycosylated and deglycosylated samples, it was possible to unambiguously identify the sites of N-linked glycosylation as well a number of N-linked glycopeptides. The O-link glycopeptides, which are present at low level ( < 1%), were not detected prior to the deglycosylation, nor could changes in peptide elution in the map following deglycosylation be correlated with potential O-linked glycosylation sites.

Application of capillary electrophoresis, high-performance liquid chromatography, on-line electrospray mass spectrometry and matrix-assisted laser desorption ionization--time of flight mass spectrometry to the characterization of single-chain plasminogen activator.

The analysis of recombinant Desmodus salivary plasminogen activator (DSPA alpha 1), a heterogeneous glycoprotein, is demonstrated through the use of high-performance liquid chromatography (HPLC), high-performance capillary electrophoresis (HPCE), liquid chromatography-electrospray mass spectrometry (LC--ES-MS), and matrix-assisted laser desorption ionization--time of flight mass spectrometry (MALDI--TOF-MS). The proteins is analyzed at three specific levels of detail: the intact protein, proteolytic digests of the protein, and fractions from the proteolytic digest. A method for "on-column" collection of HPLC fractions for subsequent transfer and analysis by HPCE and MALDI--TOF-MS is shown.

Carbohydrate structures of human tissue plasminogen activator expressed in Chinese hamster ovary cells.

Recombinant human tissue plasminogen activator (rt-PA), produced by expression in Chinese hamster ovary cells, is a fibrin-specific plasminogen activator which has been approved for clinical use in the treatment of myocardial infarction. In this study, the structures of the Asn-linked oligosaccharides of Chinese hamster ovary-expressed rt-PA have been elucidated. High mannose and hybrid oligosaccharides were released from the protein by endoglycosidase H digestion, whereas N-acetyllactosamine-type ("complex") oligosaccharides were released by peptide:N-glycosidase F digestion. The oligosaccharides were fractionated by gel permeation chromatography and anion exchange high performance liquid chromatography (HPLC), and their structures were analyzed by composition and methylation analysis, high pH anion exchange chromatography, fast atom bombardment-mass spectrometry (FAB-MS), and 500-MHz 1H NMR spectroscopy. High mannose oligosaccharides were found to account for 38% of the total carbohydrate content of rt-PA and consisted of Man5GlcNAc2, Man6GlcNAc2, and Man7GlcNAc2 in the ratio 1.8:1.7:1. Two hybrid oligosaccharides were identified and accounted for 3% of the carbohydrate of rt-PA. The N-acetyllactosamine-type oligosaccharides were found to comprise diantennary (34% of total carbohydrate), 2,4-branched triantennary (11%), 2,6-branched triantennary (9%), and tetraantennary (5%) structures. Sialylation of these oligosaccharides was by alpha (2----3) linkages to galactose. Most (greater than 90%) of the N-acetyllactosamine-type structures contained fucose alpha (1----6) linked to the Asn-linked N-acetylglucosamine residue. The distribution of oligosaccharide structures at individual glycosylation sites (Asn residues 117, 184, and 448) was also determined. rt-PA exists as two variants that differ by the presence (type I) or absence (type II) of carbohydrate at Asn-184. Tryptic glycopeptides were isolated by reversed phase high performance liquid chromatography and treated with peptide:N-glycosidase F. The oligosaccharides released from each glycosylation site were analyzed by high pH anion exchange chromatography. By this analysis, Asn-117 was demonstrated to carry exclusively high mannose oligosaccharides. When glycosylated, Asn-184 carried diantennary, 2,4-branched triantennary, 2,6-branched triantennary, and tetraantennary N- acetyllactosamine oligosaccharides in the ratio 9.0:4.5:1.4:1. Asn- 448 carried the same types of oligosaccharides, but in the ratio 7.5:1.6:2.1:1. The distributions of Asn-linked oligosaccharides at positions 117 and 448 were found not to be affected by the presence or absence of carbohydrate at position 184. The relevance of the