Mass spectrometry innovations in drug discovery and development.

This review highlights the many roles mass spectrometry plays in the discovery and development of new therapeutics by both the pharmaceutical and the biotechnology industries. Innovations in mass spectrometer source design, improvements to mass accuracy, and implementation of computer-controlled automation have accelerated the purification and characterization of compounds derived from combinatorial libraries, as well as the throughput of pharmacokinetics studies. The use of accelerator mass spectrometry, chemical reaction interface-mass spectrometry and continuous flow-isotope ratio mass spectrometry are promising alternatives for conducting mass balance studies in man. To meet the technical challenges of proteomics, discovery groups in biotechnology companies have led the way to development of instruments with greater sensitivity and mass accuracy (e.g., MALDI-TOF, ESI-Q-TOF, Ion Trap), the miniaturization of separation techniques and ion sources (e.g., capillary HPLC and nanospray), and the utilization of bioinformatics. Affinity-based methods coupled to mass spectrometry are allowing rapid and selective identification of both synthetic and biological molecules. With decreasing instrument cost and size and increasing reliability, mass spectrometers are penetrating both the manufacturing and the quality control arenas. The next generation of technologies to simplify the investigation of the complex fate of novel pharmaceutical entities in vitro and in vivo will be chip-based approaches coupled with mass spectrometry.

Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins.

We have engineered two Chinese hamster ovary cell lines secreting different recombinant glycoproteins to express high levels of human beta1,4-galactosyltransferase (GT, E.C. 2.4.1.38) and/or alpha2, 3-sialyltransferase (ST, E.C. 2.4.99.6). N-linked oligosaccharide structures synthesized by cells overexpressing the glycosyltransferases showed greater homogeneity compared with control cell lines. When GT was overexpressed, oligosaccharides terminating with GlcNAc were significantly reduced compared with controls, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. As expected, GT overexpression resulted in reduction of oligosaccharides terminating with GlcNAc, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. The more highly sialylated glycoproteins had a significantly longer mean residence time in a rabbit model of pharmacokinetics. These experiments demonstrate the feasibility of genetically engineering cell lines to produce therapeutics with desired glycosylation patterns.

The use of a porous graphitic carbon column for desalting hydrophilic peptides prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Reversed-phase high-performance liquid chromatography is the standard method for separating peptides generated from proteolytic digests of proteins. Typically, the small hydrophilic peptides from a proteolytic digest are recovered in the flowthrough fraction along with nonvolatile buffers and salts. Unfortunately, the presence of these salts can interfere with subsequent mass spectrometric analysis or Edman sequencing. To overcome this limitation, and thus enable these small peptides to be identified and characterized, we have investigated a porous graphitic carbon (PGC) column for desalting the peptides found in the unretained fraction. Using a volatile mobile phase combined with a PGC column (Hypercarb), we demonstrate that small hydrophilic peptides at the picomole level can be desalted and characterized by matrix-assisted laser desorption and ionization-time-of-flight-mass spectrometry. Even after desalting, we show that the choice of matrix still plays a significant role in distinguishing the small peptides from the matrix background. The utility of this approach is demonstrated with the flowthrough fraction of an endoproteinase Lys-C digest of a recombinant immunoglobulin. In addition, we demonstrate that a PGC column offers an alternative approach for the separation of hydrophilic, phosphorylated peptides from their unphosphorylated counterparts.

Incorporation of 15N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells.

Elevated ammonium concentrations in the medium of cultivated cells have been shown to increase the intracellular levels of uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) and uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc; Ryll et al., 1994). These sugar nucleotides are substrates for glycosyltransferases in the glycosylation pathway. In our experiments, recombinant Chinese hamster ovary cells producing an immunoadhesin glycoprotein (GP1-IgG) have been cultivated under controlled cell culture conditions in the presence of different ammonium concentrations.15N-Labeled ammonium chloride (15NH4Cl) was added exogenously to the cell culture media to determine if ammonium was incorporated into UDP-GlcNAc and cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-NANA) pools, and subsequently incorporated into GP1-IgG as N-linked glycans. The intracellular pools of UDP-activated hexosamines (UDP-GNAc) were followed during the time course of the experiment. To assess the extent of15NH4+incorporation into the glycans of GP1-IgG, the glycoprotein was first purified to homogeneity by protein A chromatography. Enzymatically released N-glycans were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. N-Glycans synthesized in the presence of15NH4Cl revealed an N-glycan-dependent increase in mass-to-charge of 2.5-4.8 Da. These results indicate that 60-70% of the total nitrogen containing monosaccharides had incorporated15N. Presumably,15NH4+was incorporated into GlcNAc and N-acetylneuraminic acid as proposed earlier (Ryll et al., 1994). This might be a universal and previously not described reaction in mammalian cells when exposed to nonphysiological but in cell culture commonly found concentrations of ammonium. The data presented here are of significance for glycoprotein production in mammalian cell culture, since it has been shown previously that elevated levels of UDP-activated hexosamines affect N-glycan characteristics such as branching and degree of amino sugar incorporation. In addition, our results demonstrate that isotope labeling in combination with MALDI-TOF-MS can be used as an alternate tool to radioactive labeling of sugar substrates in metabolic studies.

A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis.

This report describes a convenient method for the rapid and efficient release of N-linked oligosaccharides from low microgram amounts of glycoproteins. A 96-well MultiScreen assay system containing a polyvinylidene difluoride (PVDF) membrane is employed to immobilize glycoproteins for subsequent enzymatic deglycosylation. Recombinant tissue-type plasminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5 microgram rt-PA for subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The immobilization of rt-PA to the PVDF membrane did not sterically inhibit the PNGaseF-mediated release of oligosaccharides from rt-PA as determined by tryptic mapping experiments. Comparison of the oligosaccharides released from 50 micrograms of rt-PA by either the 96-well plate method or by a standard solution digestion procedure showed no significant differences in the profiles obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both neutral and sialylated oligosaccharide standards spiked into wells were recovered equally as determined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoproteins with easy removal of reagents prior to PNGaseF digestion. In addition, this method allows 60 glycoprotein samples to be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.

Analysis of acidic oligosaccharides and glycopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

2,5-Dihydroxybenzoic acid (DHB) is the most commonly used matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF) of oligosaccharides. Because acidic, sialylated oligosaccharides are detected at only the low picomole level with DHB, alternative matrices were screened to identify a matrix with a lower limit of detection. Negative-ion spectra of pure mono-, di-, and trisialylated oligosaccharides were acquired with either 6-aza-2-thiothymine or 2',4',6'-trihydroxyacetophenone (THAP) in the linear mode. Detection limits of less than 50 fmol with signal-to-noise ratios of better than 5:1 were achieved with both matrices. THAP was the preferred matrix because it provided a lower limit of detection and gave less prompt fragmentation. Incorporation of ammonium citrate into the matrix, along with vacuum drying of the sample, was required in order to obtain maximum sensitivity with THAP. No evidence of competition for ionization was found when a mixture of mono-, di-, and trisialylated oligosaccharides was analyzed with THAP. These findings suggest that MALDI/TOF analysis may provide a rapid means to identify changes in carbohydrate composition in glycoproteins. In addition, THAP offered improved sensitivity for detection of acidic glycopeptides over alpha-cyano-4-hydroxycinnamic acid.

Epitope mapping by mass spectrometry: determination of an epitope on HIV-1 IIIB p26 recognized by a monoclonal antibody.

Matrix-assisted laser desorption mass spectrometry in combination with proteolytic protection assays has been used to identify the functional epitope on HIV-1 IIIB p26 recognized by a mAb. In this procedure, the intact protein is affinity bound to an immobilized mAb under physiologic conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Protected residues were identified by matrix-assisted laser desorption mass spectrometry based on their m.w. With this approach, an 11-residue sequence was identified as the most tightly affinity-bound fragment. in addition, two less tightly bound segments were observed. These latter two residues may contain elements of a discontinuous epitope or may be residues involved in a wider contact area. The combination of matrix-assisted laser desorption and proteolytic epitope footprinting has been applied to the determination of the epitope on a recombinant protein recognized by a mAb but should be equally applicable to the definition of an epitope on a native protein in its natural folded conformation.

Monitoring cleavage of fusion proteins by matrix-assisted laser desorption ionization/mass spectrometry: recombinant HIV-1IIIB p26.

Matrix-associated laser desorption ionization/mass spectrometry (MALDI/MS) has been used to examine whole bacteria for the presence of a recombinant HIV p26 fusion protein. MALDI/MS, combined with affinity-purification techniques, is also shown to be very useful in monitoring the enzymatic cleavage of both affinity-bound fusion protein and fusion protein in solution. The combination of mass resolution, sensitivity, and speed of analysis makes MALDI/MS an attractive alternative to SDS-PAGE.

The use of high-performance anion-exchange chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to monitor and identify oligosaccharide degradation.

High-performance anion-exchange chromatography (HPAEC) with pulsed-amperometric detection was used to monitor the consistency of the oligosaccharides released from several partially purified preparations of a tissue-type plasminogen activator mutant (TNK-tPA). Differences in the oligosaccharide map were observed, primarily in the neutral carbohydrate region of the separation. Subsequent investigations using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) identified the neutral oligosaccharides to be primarily asialo-diantennary complex-type glycans with 2, 1, or 0 galactose residues. Additional asialo-triantennary and asialo-tetrantennary structures were also observed with varying amounts of galactose. Hydrolysis of the chromogenic substrate 4-methyl umbelliferyl-beta-galactoside confirmed that host-cell lysosomal beta-galactosidase is present at the first step in the purification process and is the cause of the observed glycan degradation. Subsequent steps in the purification process quantitatively remove this enzyme. Comparison of HPAEC and MALDI/TOF/MS analysis of time-course samples revealed quite similar rates of degradation and demonstrates the quantitative utility of these methods. HPAEC did not reveal significant changes in the sialylated structures as evidenced by nearly identical profiles for the sialic acid-containing structures.

Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.

Small-scale manual multiple peptide synthesis system. Application to phosphopeptide synthesis.

A system for small-scale (ca. 10-50 mumol) manual multiple peptide synthesis assembled from commercially available solid-phase extraction apparatus is described. This system was used to prepare (on a 15 mumol scale) the five monophosphorylated isomers of the peptide ASTTVSKTE, a proteolytic fragment of the C-terminal region of rhodopsin. The peptides were assembled using serine or threonine active esters without hydroxyl protection at the positions of phosphorylation. Phosphate groups were introduced using postassembly phosphitylation/oxidation according to a published procedure [Andrews, D.M., Kitchin, J. & Seale, P.W. (1991) Int. J. Peptide Protein Res. 38, 469-475; Staerkaer, G., Jakobsen, M.H., Olsen, C.E. & Holm, A., Tetrahedron Lett. 32, 5389-5392; Perich, J.W. (1992) Int. J. Peptide Protein Res. 40 134-140]. The reported system provides a relatively inexpensive approach to multiple peptide synthesis, including synthesis of phosphopeptides, for laboratories whose synthesis requirements do not justify investment in an automated multiple peptide synthesis instrument.

Mass spectrometric identification of phosphorylation sites in bleached bovine rhodopsin.

Deactivation of the visual cascade is initiated by the phosphorylation of rhodopsin. We report here identification of the two major sites of phosphorylation in bleached bovine rhodopsin using tandem mass spectrometry in conjunction with synthetic phosphopeptide standards. Both bleached and unbleached rod outer segments were cleaved with endoproteinase Asp-N to release the C-terminal fragment, residues 330-348, containing seven potential sites of phosphorylation. High-performance liquid chromatographic separation of soluble cleavage products from both unbleached and bleached rod outer segments gave a peak which was identified by tandem mass spectrometry and comparison to synthetic standards as monophosphorylated (serine 338) DDEASTTVSKTETSQVAPA. Present only in the chromatogram of bleached ROS were two peaks identified as monophosphorylated (serine 343) and diphosphorylated (serines 338 and 343) derivatives of DDEASTTVSKTETSQVAPA. These results identify serines 338 and 343 as the major sites of phosphorylation within the C-terminal region of bleached bovine rhodopsin and constitute the first example of mass spectrometric characterization of phosphorylation sites in a G-protein coupled receptor.

Matrix-assisted laser desorption mass spectrometry of rhodopsin and bacteriorhodopsin.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been used to obtain accurate molecular weight information for the integral membrane proteins bacteriorhodopsin and bovine rhodopsin desorbed from solubilized membrane preparations. Mass differences in the molecular weights measured for bleached and unbleached bacteriorhodopsin and rhodopsin indicate the removal of the retinal chromophores upon bleaching. The MALDI technique was also successful for determination of the major cleavage products obtained upon treatment of membrane bound rhodopsin with endoproteinase Asp-N and thermolysin. Our results indicate that the MALDI method is a useful means of obtaining accurate molecular weight information on hydrophobic proteins isolated in their native membranes.

Palmitylation of a G-protein coupled receptor. Direct analysis by tandem mass spectrometry.

Bovine rhodopsin has been reported to be S-palmitylated at cysteines 322 and 323 (Ovchinnikov, Y. A., Abdulaev, N. G., and Bogachuk, A.S. (1988) FEBS Lett. 230, 1-5). Using a combination of enzymatic and chemical cleavage techniques in conjunction with tandem mass spectrometry, the sites of incorporation of the palmityl groups are shown. Bovine rhodopsin in disc membranes was digested with thermolysin to generate the C-terminal fragment (241-327), which was subsequently cleaved with cyanogen bromide to generate the peptide Val-Thr-Thr-Leu-Cys-Cys-Gly-Lys-Asn-Pro (318-327). A bis-S-palmitylated synthetic standard had the same retention time by reversed-phase high performance liquid chromatography as the isolated peptide and the same molecular weight (MH+1511.7) by liquid secondary ion mass spectrometry. Dithiothreitol reduction of both the isolated and the synthetic peptide cleaved the two thioester-linked palmityl groups to produce reduction products of the same appropriately decreased molecular weight (MH+1035.5). Tandem mass spectrometry of the isolated and the synthetic peptide identified the sites of attachment of the palmityl groups on cysteines 322 and 323. These results prove the modification of cysteines 322 and 323 with palmitic acid in bovine rhodopsin, and illustrate the utility of mass spectrometry to characterize the post-translational modifications in G-protein coupled receptors.

Measurement of lysergic acid diethylamide (LSD) in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry.

A previously reported procedure for quantification of LSD in urine was modified to permit measurement of the drug in plasma. After addition of deuterium-labelled LSD, the plasma is extracted and the extract is treated with trifluoroacetylimidazole to convert the LSD to its N-trifluoroacetyl derivative. The derivatized LSD is analyzed by capillary column gas chromatography/negative ion chemical ionization. Plasma fortified with known concentrations of LSD gave linear responses from 0.1 to 3.0 ng/mL with this assay. The method was used to determine pharmacokinetic parameters for LSD after oral administration (1 microgram/kg) to a male volunteer. The apparent plasma half-life was determined to be 5.1 h. The peak plasma concentration of 1.9 ng/mL occurred 3 h after administration.

N-benzylimidazole, a high magnitude inducer of rat hepatic cytochrome P-450 exhibiting both polycyclic aromatic hydrocarbon- and phenobarbital-type induction of phase I and phase II drug-metabolizing enzymes.

The effects of N-benzylimidazole on hepatic microsomal and cytosolic drug-metabolizing enzymes were compared to the effects produced by phenobarbital, beta-naphthoflavone, a polycyclic aromatic hydrocarbon, and Aroclor 1254, a polychlorinated biphenyl mixture. N-Benzylimidazole was a "high magnitude" inducer of male rat hepatic cytochrome P-450, inducing cytochrome P-450 over 3 times above control. N-Benzylimidazole exhibited mixed type induction of cytochrome P-450, producing both polycyclic aromatic hydrocarbon- and phenobarbital-type induction. There was no evidence of imidazole (isoniazid) type induction characteristics. Microsomes from rats treated with either Aroclor 1254 or N-benzylimidazole showed a common pattern of induction of the cytochrome P-450-dependent properties and glucuronosyltransferase activities, and the electrophoretic profiles of proteins were also similar. Cytosolic glutathione transferase activity was also induced similarly after treatment with the two agents.