Aspects of oligonucleotide and peptide sequencing with MALDI and electrospray mass spectrometry

Biopolymer sequencing with mass spectrometry has become increasingly important and accessible with the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Here we examine the use of sequential digestion for the rapid identification of proteolytic fragments, in turn highlighting the general utility of enzymatic MALDI ladder sequencing and ESI tandem mass spectrometry. Analyses were performed on oligonucleotides ranging in size from 2 to 50 residues, on peptides ranging in size from 7 to 44 residues and on viral coat proteins. MALDI ladder sequencing using exonuclease digestion generated a uniform distribution of ions and provided complete sequence information on the oligonucleotides 2-30 nucleic acid residues long. Only partial sequence information was obtained on the longer oligonucleotides. C-terminal peptide ladder sequencing typically provided information from 4 to 7 amino acids into the peptide. Sequential digestion, or endoprotease followed by exoprotease exposure, was also successfully applied to a trypsin digest of viral proteins. Analysis of ladder sequenced peptides by LCMS generated less information than in the MALDI-MS analysis and ESI-MS2 normally provided partial sequence information on both the small oligonucleotides and peptides. In general, MALDI ladder sequencing offered information on a broader mass range of biopolymers than ESI-MS2 and was relatively straightforward to interpret, especially for oligonucleotides.     

Capillary electrophoresis combined with matrix-assisted laser desorption/ionization mass spectrometry; continuous sample deposition on a matrix-precoated membrane target

Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) were combined in an off-line arrangement to provide separation and mass analysis of peptide and protein mixtures in the attomole range. A membrane target, precoated with MALDI matrix, was used for the continuous deposition of effluent exiting from a CE device. A sample track was produced by linear movement of the target during the electrophoretic separation and this track was subsequently analyzed by MALDI/MS. The technique is effective for peptides and proteins, having limits of detection (signal-to-noise >3) of about 50 amol for neurotensin (1673 Da) and 250 amol for cytochrome c (12361 Da) and apomyoglobin (16951 Da). The electrophoretic separation achieved from the membrane target, as measured by theoretical plate numbers from the mass spectrometric data, can be as high as 80-90% of that achieved by on-line UV detection under optimal conditions, although band broadening occurs and with some loss of separation efficiency. Non-volatile buffers such as 10-50 mM phosphate can also be used in the electrophoresis process and directly deposited on the membrane. The use of post-source decay techniques is shown for peptides in the CE sample track in order to obtain sequence verification. The effectiveness of this method of integration of CE and MALDI/MS is demonstrated with both peptide and protein mixtures and with the analysis of a tryptic digest of a protein.     

Characterization of serine and threonine phosphorylation sites in beta-elimination/ethanethiol addition-modified proteins by electrospray tandem mass spectrometry and database searching

A new method for the characterization of serine and threonine phosphorylation sites in proteins has been developed. After modification of a phosphoprotein by beta-elimination/ethanethiol addition and conversion of phosphoserine and phosphothreonine residues to S-ethylcysteinyl or beta-methyl-S-ethylcysteinyl residues, the modified protein was subjected to proteolytic digestion. Resulting digests were analyzed by a combination of microbore liquid chromatography, electrospray ionization tandem (MS/MS) ion trap mass spectrometry and database searching to identify original phosphorylated residues. The computer program utilized (SEQUEST) is capable of identifying peptides and modified residues from uninterpreted MS/MS spectra, and using this method, all of the five known phosphorylation sites in bovine beta-casein were identified. Application of the method to multiply phosphorylated human high molecular weight neurofilament protein (NF-H) resulted in the identification of 21 peptides and their modified residues and hence, the in vivo phosphorylation sites. These included 26 KSP and 1 KTP site, all of which occur in the KSP repeat C-terminal tail domain (residues 502-823). One site at residue 518 was previously uncharacterized. A novel non-KSP serine at residue 421 near the KLLEGEE region in a IPFSLPE motif was characterized as phosphorylated (or glycosylated). The 27 characterized phosphorylation sites occur at S/TP residues in the following motifs: KSPVKEE, KSPAEAK, KSPEKEE, KSPAEVK, KSPEKAK, KSPPEAK, KSPVKAE, and KTPAKEE. On the basis of kinase consensus sequences, all of these motifs, including the previously unreported KTPAKEE motif, can be phosphorylated by proline-directed kinases. Advantages of the new method vis-a-vis our previously reported method [Jaffe, H., Veeranna, Shetty, K. T., and Pant, H. C. (1998) Biochemistry 37, 3931-3940] include (i) production of diastereomers eluting at different retention times increased the chances of peptide identification, (ii) increased hydrophobicity and hence retention time of the modified peptides, (iii) facilitation of positive ion production, and (iv) increased susceptibility to tryptic digestion as a result of conversion of negatively charged phosphorylated residues to neutral S-ethylcysteine or beta-methyl-S-ethylcysteine residues.     

Characterization of pathotype-specific epitopes of newcastle disease virus fusion glycoproteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and post-source decay sequencing

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) was used to characterize the F2 polypeptide of the fusion (F) protein of an avirulent isolate (VRI 82-6409) of Newcastle disease virus (NDV) that was previously identified by immunochemical screening as having a variant cleavage activation sequence in its fusion protein precursor (F0). The major glycoform of the intact F2 polypeptide of the VRI 82-6409 isolate was 89 Da smaller than the F2 polypeptide of the avirulent V4 isolate of the Queensland strain of NDV. Analysis of AspN protease digests of the F2 polypeptides by MALDI/TOF-MS, with and without high-performance liquid chromatographic (HPLC) separation, showed this mass difference to be due to a combination of differences in the extents of glycosylation and an amino acid difference in the AspN peptides derived from the C-termini of the F2 polypeptides. Accuracies achieved in analysis of the AspN peptides allowed the identification of this amino acid difference as glutamic acid in the VRI 82-6409 isolate compared with glycine in the V4 isolate. Analysis of fragments formed by post-source decay (PSD) of ions of the C-terminal AspN peptides localized the difference to the C-terminal residues of the respective F2 polypeptides. The present study demonstrated that MALDI/TOF-MS is a highly effective technique for the characterization of NDV variants identified by immunochemical screening of pathotype-specific epitopes at the C-termini of their F2 polypeptides.     

Matrix-assisted laser desorption/ionization mass spectrometry of noncovalent protein-transition metal ion complexes

Transition metal ion complexes with proteins and peptides are important in many areas of analytical and biological chemistry. We used positive and negative ion MALDI-MS to detect complexes with Cu and Ni ions, and show that the specific and non-specific transition metal ion-peptide complexes can be distinguished by the use of different analytical protocols. The pH dependent stability of these complexes is also reflected in the MALDI data. We further show that triple complexes of peptides or protein with chelated metal ions can be detected efficiently and rapidly by MALDI mass spectrometry. Such triple complexes play an important role in metal chelate affinity chromatography, where histidine containing biopolymers in particular are thought to bind metal-ligand complexes, depending on the oxidation state of the metal and the number of unoccupied coordination sites of the ligand.     

Post-translational modifications of water-soluble human lens crystallins from young adults

Post-translational modifications of the water-soluble human lens crystallins from young adult donors were identified and located using electrospray ionization mass spectrometric analysis of the intact proteins and fast atom bombardment mass spectrometry of enzymatic digests. Peptides corresponding to all of the sequences of alpha A-, alpha B-, and beta B2-crystallins were found, permitting the entire sequences to be searched for modifications. The major portions of these three crystallins were not modified. Modifications of alpha A-crystallin that were detected included 2 phosphorylated Ser residues (1 of which appears to be unique to human lenses), deamidation at some Gln and Asn residues, a disulfide bond between Cys-131 and Cys-142, and loss of the COOH-terminal Ser residue. Three phosphorylated Ser residues, but no deamidation, were found in alpha B-crystallin. The molecular weights of neither the intact protein nor the peptides in the enzymatic digests indicated any post-translational modification of the principal beta-crystallin, beta B2. The molecular weights of the other beta- and gamma-crystallins for which sequences have been published suggested the presence of post-translational modifications or errors in the published sequences. Although enough peptides were found to establish the presence of specific proteins, peptides corresponding to all portions of these proteins were not found, and elucidation of these structures is not yet complete. This mass spectrometric characterization of the total water-soluble proteins from normal young adult lenses provides a reference data base for future investigations of the modifications present in aged and cataractous lenses.     

Reduced chaperone-like activity of alpha A(ins)-crystallin, an alternative splicing product containing a large insert peptide

alpha-Crystallin is a multimeric protein complex which is constitutively expressed at high levels in the vertebrate eye lens, where it serves a structural role, and at low levels in several non-lenticular tissues. Like other members of the small heat shock protein family, alpha-crystallin has a chaperone-like activity in suppressing nonspecific aggregation of denaturing proteins in vitro. Apart from the major alpha A- and alpha B-subunits, alpha-crystallin of rodents contains an additional minor subunit resulting from alternative splicing, alpha A(ins)-crystallin. This polypeptide is identical to normal alpha A-crystallin except for an insert peptide of 23 residues. To explore the structural and functional consequences of this insertion, we have expressed rat alpha A- and alpha A(ins)-crystallin in Escherichia coli. The multimeric particles formed by alpha A(ins) are larger and more disperse than those of alpha A, but they are native-like and display a similar thermostability and morphology, as revealed by gel permeation chromatography, tryptophan fluorescence measurements, and electron microscopy. However, as compared with alpha A, the alpha A(ins)-particles display a diminished chaperone-like activity in the protection of heat-induced aggregation of beta low-crystallin. Our experiments indicate that alpha A(ins)-multimers have a 3-4-fold reduced substrate binding capacity, which might be correlated to their increased particle size and to a shielding of binding sites by the insert peptides. The structure-function relationship of the natural mutant alpha A(ins)-crystallin may shed light on the mechanism of chaperone-like activity displayed by all small heat shock proteins.     

Inhibition of the self-assembly of collagen I into fibrils with synthetic peptides. Demonstration that assembly is driven by specific binding sites on the monomers

A series of experiments were carried out to test the hypothesis that the self-assembly of collagen I monomers into fibrils depends on the interactions of specific binding sites in different regions of the monomer. Six synthetic peptides were prepared with sequences found either in the collagen triple helix or in the N- or C-telopeptides of collagen I. The four peptides with sequences found in the telopeptides were found to inhibit self-assembly of collagen I in a purified in vitro system. At concentrations of 2.5 mM, peptides with sequences in the C-telopeptides of the alpha1(I) and alpha2(I) chain inhibited assembly at about 95%. The addition of the peptide with the alpha2-telopeptide sequence was effective in inhibiting assembly if added during the lag phase and early propagation phase but not later in the assembly process. Experiments with biotinylated peptides indicated that both the N- and C-telopeptides bound to a region between amino acid 776 and 822 of the alpha(I) chain. A fragment of nine amino acids with sequences in the alpha2-telopeptide was effective in inhibiting fibril assembly. Mutating two aspartates in the 9-mer peptide to serine had no effect on inhibition of fibril assembly, but mutating two tyrosine residues and one phenylalanine residue abolished the inhibitory action. Molecular modeling of the binding sites demonstrated favorable hydrophobic and electrostatic interactions between the alpha2telopeptide and residues 781-794 of the alpha(I) chain.     

Evidence for the participation of alpha B-crystallin in human age-related nuclear cataract

The aim of this study was to determine if the unusual coloured species characteristic of age-related nuclear cataract could be localised to specific residues of the crystallins. The insoluble, crosslinked and coloured cataract protein fraction (CPF) was isolated from cataract human lenses. Using a combination of tryptic digestion, gel filtration and multiple reversed phase high performance liquid chromatography (RP-HPLC), coloured peaks were isolated and subjected to amino acid sequence analysis. With these techniques, it was hoped to identify and locate the modified residues. Sequence information was obtained on 16 'coloured' peptides. Many of the peptides were found to be derived from alpha B-crystallin. When redundancies are taken into account, six distinctive peptides were found to be derived from alpha B-crystallin; one from beta B1-crystallin, two from beta A3/A1-crystallin and three from gamma S-crystallin. Three sites of possible crystallin residue isomerisation to modification were detected in the alpha B- and beta A3/beta A1-crystallins, including probable asp isomerisation at residues 25 and 36 in alpha B-crystallin. Since the CPF is unique to nuclear cataract lenses, these data suggest that alpha-crystallin, and alpha B-crystallin in particular, may be implicated in the cataract process. This finding supports that of a recent study on cataract proteins using pronase digestion [Chen YC, Reid GE, Simpson RJ, Truscott RJW. Exp Eye Res 1997;65:835.]     

Chemical modifications and dissociation characteristics of tyrosine and tryptophan residues in alpha-crystallin

A quantitative estimation of surface accessibility of aromatic residues in alpha-crystallin from goat lens has been accomplished by chemical modifications using different specific reagents having varying sizes. Results of modification of tyrosine residues with N-acetylimidazole and tetranitromethane when combined with those of ionization studies carried out with hydroxyl ions having the smallest size reveal different classes of tyrosine residues in the native protein: 78 +/- 2 residues have been found to be easily available for modification; among the rest, 94 +/- 2 residues appear to be comparatively less exposed to the reagents while 28 +/- 2 residues are found to be completely unavailable for modification in the native protein and are modified only when the protein is denatured. Modification of tryptophan residues with H2O2 also indicates different classes of these residues available for oxidation at different concentrations of the oxidant. 34 +/- 2 residues of tryptophan are found to be easily oxidized at a lower concentration of H2O2 during the first phase of the reaction. The remaining tryptophan residues appear to be less exposed to the reagent. This is also corroborated from the studies of reactivities of these residues towards another specific but bulkier reagent, 2-hydroxy-5-nitrobenzyl bromide. These surface exposed aromatic residues in alpha-crystallin may be considered to be vulnerable to in vivo oxidative modifications forming insoluble aggregates which may finally contribute to the formation of cataract.     

Analysis of variant forms of porcine surfactant polypeptide-C by nano-electrospray mass spectrometry

Electrospray (ES) mass spectrometry has been used to analyse preparations of porcine pulmonary surfactant polypeptide-C (SP-C). A number of variant forms of the native 35-residue dipalmitoylated peptide were detected including (a) C-terminally methylated SP-C, (b) C-terminally methylated and methionine oxidized SP-C, (c) N-terminally truncated, C-terminally methylated and methionine oxidized SP-c, (d) C-terminally elongated, C-terminally methylated and methionine oxidized SP-C, and (e) tripalmitoylated, C-terminally methylated and methionine oxidized SP-C. C-terminal methylation and methionine oxidation are probably a consequence of the sample handling procedure. The occurrence of the C-terminally elongated form of SP-C has implications for the in vivo processing of proSP-C and the Tandem mass spectrometry (MS/MS) was used to confirm the amino acid sequence of SP-C and the presence of palmitoyl groups covalently linked to the peptide. Some of the structures of the variant forms of SP-C were determined by MS/MS.     

Identification and characterization of glycosylation sites in human serum clusterin

Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 +/- 250 and 63,507 +/- 200). Mass spectrometry also revealed molecular heterogeneity associated with both the alpha and beta subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17-27% carbohydrate by weight, the alpha subunit contains 0-30% carbohydrate and the beta subunit contains 27-30% carbohydrate. Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N-linked glycosylation sites were identified, three in the alpha subunit (alpha 64N, alpha 81N, alpha 123N) and three in the beta subunit (beta 64N, beta 127N, and beta 147N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site beta 64N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site beta 147N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennary with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated alpha and beta subunits, including all Ser- and Thr-containing peptides. No evidence was found for the presence of O-linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure-function relationships and the role clusterin glycosylation plays in physiological function.     

Improved capillary electrophoretic separation of glycosylated oligopeptides through addition of poly(vinyl alcohol), and analysis by electrospray mass spectrometry

A method for the analysis of O-glycosylation of peptides has been developed, combining capillary electrophoretic (CE) separation and electrospray ionization mass spectrometry. Synthetic peptides with apomucin 'tandem repeat' sequences which present potential O-glycosylation sites on threonine and serine residues were used as model system. In vitro O-glycosylated peptide samples were obtained by incubation of the peptides with human gastric microsomal homogenates containing N-acetylgalactosamine transferase activity in the presence of uridyl diphosphate N-acetylgalactosamine (UDP-GalNAc). CE was carried out in the presence of the linear polymer poly(vinyl alcohol) in the electrophoresis solvent, resulting in a greatly improved separation of the up to five different glycoforms of peptides with lengths of 8, 16 or 23 amino acids, and the unglycosylated peptides. After separation and peak collection, the number of modifications with N-acetyl galactosamine (GalNAc) could be determined by electrospray ionization mass spectrometry. The glycosylation pattern was shown to depend on the amino acid sequence of the peptides.     

Identification of 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid binding sequences in alpha-crystallin

The hydrophobic binding sites in alpha-crystallin were evaluated using fluorescent probes 1,1'-bi(4-anilino)naphthalenesulfonic acid (bis-ANS), 8-anilino-1-naphthalene sulfonate (ANS), and 1-azidonaphthalene 5-sulfonate (1,5-AZNS). The photolysis of bis-ANS-alpha-crystallin complex resulted in incorporation of the probe to both alphaA- and alphaB-subunits. Prior binding of denatured alcohol dehydrogenase to alpha-crystallin significantly decreased the photoincorporation of bis-ANS to alpha-crystallin. Localization of bis-ANS incorporated into alphaA-crystallin resulted in the identification of residues QSLFR and HFSPEDLTVK as the fluorophore binding regions. In alphaB-crystallin, sequences DRFSVNLNVK and VLGDVIEVHGK were found to be the bis-ANS binding regions. Of the bis-ANS binding sequences, HFSPEDLTVK of alphaA-crystallin and DRFSVNLNVK and VLGDVIEVHGK of alphaB-crystallin were earlier identified as part of the sequences involved in their interaction with target proteins during the molecular chaperone-like action. The hydrophobic probe, 1,5-AZNS, also interacted with both subunits of alpha-crystallin. Localization of 1,5-AZNS binding site in alphaB-crystallin lead to the identification of HFSPEEK sequence as the interacting site in this subunit of alpha-crystallin. Glycated alpha-crystallin displayed decreased ANS fluorescence and loss of chaperone-like function, suggesting the involvement of glycation site as well as ANS binding site in chaperone-like activity display.     

Membrane interaction of synthetic peptides related to the putative fusogenic region of PH-30 alpha, a protein in sperm-egg fusion

In order to investigate the relationship between structure and function of a putative fusogenic region of PH-30a, a protein active in sperm-egg fusion, two peptides, SFP22 and SFP23, whose sequences correspond to the residues 90-111 and 89-111 of PH-30 alpha, respectively, were chemically synthesized. An analog of SFP23, SFP23AA, which has an Ala-Ala sequence instead of the Pro-Pro sequence in SFP23, was also prepared. The CD study indicated that SFP22 and SFP23 mainly took a beta-structure in the presence of DPPC and DPPC/DPPG (3/1) vesicles, while SFP23AA showed an alpha-helical pattern though the alpha-helical content calculated was low (25-30%). alpha-Helical CD curve was observed for these peptides in trifluoroethanol. The membrane-perturbing activity of SFP22 and SFP23 was weaker than that of SFP23AA. On the other hand, the membrane-fusogenic activity of SFP22 and SFP23 to acidic phospholipid bilayers was much stronger than that of SFP23AA. All the peptides caused very weak cell lysis. These results are consistent with the reported speculation [Blobel, C. P. et al. (1992), Nature (London) 356, 248-252] that residues 90-111 of PH-30 alpha may be the fusogenic region and suggest that the Pro-Pro sequence is one of the important factors for holding the active secondary structure of the fusogenic region of PH-30 alpha in membranes.     

Negative ion postsource decay time-of-flight mass spectrometry of peptides containing acidic amino acid residues

Acidic peptides have been studied by negative ion postsource decay (PSD) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The peptides contained from 5 to 16 residues and were chosen on the basis of their patterns of the acidic residues. Using typical MALDI sample preparation techniques employing an acidic matrix, gastrin I (1-14), and epidermal mitosis inhibiting pentapeptide yielded much larger deprotonated ion signals, [M - H]-, than protonated ions, [M + H]+. This may be due to their absence of basic residues, coupled with their arrays of acidic residues. The PSD fragmentation of the peptide negative ions showed that an array of acidic residues, as in gastrin I (1-14), yielded simple spectra containing mainly backbone cleavage ions from the C-terminus. Hirudin (54-65), which contains two sets of two consecutive Glu residues, and fibrinopeptide A and fibrinopeptide B, with isolated acidic residues, also showed backbone cleavages as common fragment ions. In addition, the two sets of isolated consecutive amino acid residues in Cys(Bzl)84-CD4 (81-92) and hirudin (54-56) yielded internal ions from the cleavages at the (O=C)-NH bond between the acidic residues. Also observed were ions with unique side chain losses, such as the loss of C6H4O from a tyrosine residue and SCH2C6H5 and CH2C6H5 from a benzylated cysteine residue. Compared to the positive mode, the negative-ion PSD yielded fewer fragments which usually involved only one type of backbone cleavage (e.g., [Yn - H2O]-). These simple spectra aided interpretation. Overall, the acidic peptides studied yielded negative ion PSD spectra that were useful for peptide sequencing.     

Purification and characterization of plantaricin A, a Lactobacillus plantarum bacteriocin whose activity depends on the action of two peptides

A Lactobacillus plantarum bacteriocin, plantaricin A, has been purified to homogeneity by ammonium sulphate precipitation, binding to cation exchanger and Octyl-Sepharose, and reverse-phase chromatography. The bacteriocin activity was associated with two peptides, termed alpha and beta, which were separated upon reverse-phase chromatography. Bacteriocin activity required the complementary action of both the alpha and beta peptides. From the N-terminal end, 21 and 22 amino acid residues of alpha and beta, respectively, were sequenced. Further attempts at sequencing revealed no additional amino acid residues, suggesting that either the C terminus had been reached or that modifications in the next amino acid residue blocked the sequencing reaction. Judging from their amino acid sequence, alpha and beta may be encoded by the same gene, since alpha appeared to be a truncated form of beta. Alanine, the first amino acid residue at the N-terminal end of beta was not present at this position in alpha. Otherwise the sequences of alpha and beta appeared to be identical. The calculated molecular masses of the sequenced part of alpha and beta were 2426 and 2497 Da, respectively. The molecular masses of alpha and beta as determined by mass spectroscopy were 2687 +/- 30 and 2758 +/- 30 Da, respectively, indicating that (i) the only difference between alpha and beta was the presence of the N-terminal alanine residue in beta, and that (ii) in addition to the sequenced residues, two to three unidentified amino acid residues are present at the C-terminal ends of the alpha and beta peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the microheterogeneities of PIXY321, a genetically engineered granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein expressed in yeast

PIXY321, a human cytokine analog genetically engineered by the fusion of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), was expressed in yeast under the control of the alcohol dehydrogenase 2 (ADH2) promoter and the alpha-mating factor expression system. To provide the material necessary for the evaluation of PIXY321 in clinical trials, the production was scaled up to the 1200-1 scale and the PIXY321 molecule isolated by four successive steps of ion-exchange chromatography. Multiple heterogeneities, due to the presence of different patterns of glycosylation as well as multiple amino acid sequences at both N and C termini, were characterized on the purified molecule using complementary analytical techniques including electrophoresis, liquid chromatography and electrospray mass spectrometry. Four different N-terminal sequences were identified but simplified to a reproducible ratio of two sequences, the mature form and a form starting at Ala3, by adjustment of the process conditions. Molecules lacking 1-6 residues at the C-terminus were identified and their relative frequencies quantified. Amino acid modifications, such as three oxidized Met residues at positions 79, 141 and 187 and one deamidated Asn residue at position 176, were detected at low level. Microheterogeneities in glycosylation were characterized on four different sites, one located in the GM-CSF portion and three in the IL-3 portion of the molecule. The sites were shown to be differentially occupied and to carry 0-10 mannose residues according to their location in the sequence. Precise measurement of the heterogeneities at the molecular level were used to tune the process conditions and ensure reproducibility of the clinical product between lots.     

Structures of bacitracin A and isolated congeners: sequencing of cyclic peptides with blocked linear side chains by electrospray ionization mass spectrometry

The bacitracin antibiotic complex consists principally of bacitracin A, a peptide antibiotic containing seven amino acid residues in a ring and five amino acid residues in a blocked side chain, together with a mixture of minor components presumably related but of unknown structures. A preparative high-performance liquid chromatographic method was developed for isolating the minor components A2, B1 and B2 which were then characterized by amino acid analysis, exact mass fast atom bombardment (FAB) mass spectrometry, FAB tandem mass spectrometry (MS/MS) and electrospray ionization (ESI) mass spectrometry. For bacitracins A (MW 1421), A2 (MW 1421), B1a (MW 1407), B1b (MW 1407), B2 (MW 1407) and F (MW 1419), the side chain sequences were determined by ESI MS/MS and ESI nozzle-skimmer collision-induced dissociation (CID) mass spectrometry and the ring sequences elucidated by ESI nozzle-skimmer CID MS/MS. Relative to bacitracin A, bacitracin A2a has the modified isoleucine residue at position 1 replaced by a modified allo-isoleucine residue, bacitracin B1a has the isoleucine residue at position 8 replaced by a valine residue, bacitracin B1b has the isoleucine residue at position 5 replaced by a valine residue and bacitracin B2 has the modified isoleucine residue at position 1 replaced by a modified valine residue. FAB tandem mass spectra were shown to be consistent with the above structural assignments for the isolated bacitracin components. Structures were also proposed for the trace bacitracin components C1 (MW 1393) and D1 (MW 1379) using ESI MS/MS data obtained from the analysis of the bacitracin complex without isolation.     

Identification and characterization of posttranslational modifications of proteins by MALDI ion trap mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometry is shown to be a powerful tool for the elucidation of protein modifications. Low-energy covalent bonds that originate from certain posttranslational modifications dissociate preferentially to produce characteristic mass spectrometric signatures that prove useful for the accurate, confident identification and characterization of such modifications. Because the MALDI ion trap is an authentic tandem mass spectrometer, it proves feasible to acquire secondary information to test hypotheses as to the nature and site of the putative modifications--further increasing the reliability of the tool. The method combines the advantageous features of MALDI (i.e., the ability to measure the same sample repeatedly, to measure unfractionated complex mixtures without the need for sample cleaning, and to determine peptide mixtures with subpicomole sensitivity) with the ease and the speed of the ion trap measurement. We demonstrate how the unique properties of MALDI ion trap MS can be used to address problems involving the determination of both native posttranslational modifications of proteins (e.g., disulfide mapping, glycosylation determination, and phosphorylation determination) and non-native chemical modifications of proteins (e.g., methionine oxidation and photo-cross-linking of proteins with DNA).