Structure elucidation of a purple peptide found during the purification of a recombinant protein from Escherichia coli.

A purple substance (4) partially co-purified with a recombinant human B-type natriuretic peptide (hBNP), following an E. coli fermentation. The structure of the compound was elucidated by NMR, electrospray and FAB mass spectrometry. The chromophore is a 1,4-naphthoquinone condensed with the N-terminal cysteine of a heptapeptide by its NH2- and SH-groups to form a dihydro-thiazine ring. The peptide sequence was determined as Cys-Lys-Val-Leu-Arg-Arg-His by mass spectrometric techniques. CID and data base matching identified it as the C-terminus of the 32-amino-acid recombinant peptide hBNP. This modification of an N-terminal Cys may be a more general phenomenon with implications for the production of heterologous proteins by microorganisms.

Application of capillary high-performance liquid chromatography to biotechnology, with reference to the analysis of recombinant DNA-derived human growth hormone.

Using capillary HPLC, femtomole amounts of recombinant DNA-derived human growth hormone (rhGH) have been successfully detected from solutions at nanomolar concentrations. The separation used capillaries of 15 cm x 320 microns I.D. and detection was with a UV absorbance detector containing a capillary Z-shaped flow-cell. A sample of rhGH that was recovered from rat serum was analyzed by capillary reversed-phase HPLC, using both acidic- and neutral-pH mobile phases, as well as by capillary ion-exchange chromatography. When compared to HPLC separations performed at flow-rates of 1 ml/min, the sensitivity of the detection was increased 200 times, without any loss in resolution. Sub-microgram amounts of rhGH were also analyzed by tryptic mapping using capillary HPLC and peptides were identified by capillary LC-MS.

Tissue plasminogen activator has an O-linked fucose attached to threonine-61 in the epidermal growth factor domain.

An unusual type of glycosylation has been observed for tissue plasminogen activator (t-PA). The monosaccharide fucose is glycosidically linked to threonine-61 in the epidermal growth factor region of t-PA. The presence of O-linked fucose was demonstrated by carbohydrate analysis and mass spectrometry of tryptic and chymotryptic peptides that contain this site. The susceptibility of the fucose residue to alpha-fucosidase indicated that it was in the alpha-anomeric configuration. Fucosylation of threonine-61 was observed in t-PA isolated from the Bowes melanoma cell line and from recombinant expression systems using Chinese hamster ovary or human embryonic kidney cells. Fucosylation of the homologous residue in prourokinase has also been reported recently. Our results indicate that this novel type of glycosylation may be common to the epidermal growth factor domains found in coagulation and fibrinolytic proteins and, therefore, suggest that the modification may have functional significance.

Characterization of humanized anti-TAC, an antibody directed against the interleukin 2 receptor, using electrospray ionization mass spectrometry by direct infusion, LC/MS, and MS/MS.

Characterization of a humanized monoclonal antibody (Hu-anti-TAC) directed against a surface protein expressed on T-lymphocytes was performed with an electrospray mass spectrometer. Capillary reversed-phase liquid chromatography (LC)/mass spectrometry (MS) and direct infusion MS were utilized along with tandem MS/MS analysis to confirm the sequence and to determine the sources of heterogeneity in Hu-anti-TAC. The MS analysis was performed on disulfide-reduced and trypsin-digested samples of the antibody. Two forms of diantennary carbohydrate structures were identified and found to be consistent with those reported for the human IgG1 framework. The analysis demonstrated that the N-terminus was modified by conversion of a glutamine residue to pyroglutamic acid. Another source of heterogeneity was the partial removal of the C-terminal lysine residue and was confirmed by mass calculations of tryptic peptides followed by MS/MS sequencing. This study demonstrates that the high sensitivity of electrospray mass spectrometry when combined with capillary chromatography can allow detailed characterization of microgram samples of high molecular weight proteins such as antibodies.

Deamidation and isoaspartate formation during in vitro aging of recombinant tissue plasminogen activator.

When incubated at pH 7.3, 37 degrees C, human recombinant tissue plasminogen activator accumulated 0.77 mol of isoaspartate per mol of plasminogen activator over a 14-day period. Isoaspartate was detected by enzymatic transfer of 3H-labeled methyl groups from S-adenosyl-L-methionine in a reaction catalyzed by protein L-isoaspartyl methyltransferase. Analysis of tryptic peptides derived from aged plasminogen activator revealed that the two major sites of isoaspartate accumulation resulted from deamidation of Asn58 in the sequence -FNGG- and Asn177 in the sequence -GNSD-. Significant levels of isoaspartate also accumulated via deamidation of Asn37 in the sequence -CNSG-. All three sites occur in sequences predicted from studies with synthetic peptide to be unstable. All three sites appear to be on the surface of the protein, and all three occur in regions of the protein predicted to have higher than average chain mobility. These findings add support to the idea that sequence and flexibility play major roles in determining susceptibility to deamidation and peptide bond isomerization at Asn and Asp sites under mild conditions. These studies also illustrate the utility of enzymatic methylation for characterizing sites of deamidation in a large protein that contains numerous disulfide bonds and several sites of glycosylation.

The type II isoform of bovine brain protein L-isoaspartyl methyltransferase has an endoplasmic reticulum retention signal (...RDEL) at its C-terminus.

Bovine brain is known to contain two major isoforms of protein L-isoaspartyl methyltransferase (PIMT), an enzyme that facilitates repair of atypical L-isoaspartyl peptide bonds in proteins. Although the two isoforms can be separated by anion-exchange chromatography, they appear to have similar, if not identical, substrate specificities in vitro. The more basic type I isoform has been extensively characterized, and its complete sequence has been reported. The present study was undertaken in an attempt to understand the structural and functional uniqueness of the more acidic type II isoform. Electrospray mass spectrometry of the intact enzymes revealed that the type II isoform is approximately 43 amu heavier than the type I isoform. Cyanogen bromide cleavage followed by HPLC with on-line mass analysis revealed that the type II isoform contains a unique C-terminal fragment which is 43 amu heavier than the corresponding fragment from the type I isoform. Amino acid composition analysis and direct sequencing of this fragment indicate that the type II isoform ends in the sequence ...RDEL, while the type I is known to end in ...RWK. Since ...RDEL, like ...KDEL, serves as an effective endoplasmic reticulum retention signal, we propose that the type II isoform serves to repair damaged proteins within the endoplasmic reticulum or, perhaps, within some other specialized compartment of the cell. Comparison of the protein sequences of the two bovine brain isoforms to DNA sequences for rodent PIMT reported by others suggests that the type II isoform may be produced by splicing within the codon for Arg224.

Identification of carbohydrate structures in glycoprotein peptide maps by the use of LC/MS with selected ion extraction with special reference to tissue plasminogen activator and a glycosylation variant produced by site directed mutagenesis.

Electrospray ionization mass spectrometry utilizing a single quadrupole on line with reversed-phase HPLC (LC/MS) enables the characterization of glycoproteins in a relatively short period of time. In this approach the protein is digested with a suitable protease and the peptides are separated by reversed-phase HPLC and detected by electrospray ionization mass spectrometry. The glycopeptides are initially observed as a cluster of negatively sloping ions in a contour plot of data from the LC/MS run (m/z vs retention time) or as a characteristics series of masses at different elution times. The search for a particular glycopeptide is based on previously known carbohydrate structures and on consensus glycosylation sites. Further structural information is obtainable with glycosidase digestion and LC/MS analysis. The mass shifts following glycosidase digestion allow further confirmation of the structure. This approach identifies the site of attachment of two hybrid glycoforms to the T11 tryptic peptide in a reversed-phase tryptic map of recombinant tissue plasminogen activator (rt-PA). Use of selected ion extraction of the LC/MS data files allows one to graphically describe the elution order of closely related glycopeptides. The potential of LC/MS for the characterization of small amounts of unknown glycoproteins is shown by the study of an rt-PA mutant. A new potential site for glycosylation is created by site directed mutagenesis of wild type rt-PA with replacement of a threonine residue with asparagine at residue 103. An examination of a tryptic map shows that the mutant contains two new complex carbohydrate chains. The introduction of the new asparagine proximal to asparagine 117 changes this native high-mannose site in rt-PA to a complex-type glycosylation. This method allows rapid identification of carbohydrate containing peptides and yields useful structural information on microgram amounts of material.

Processing of beta-amyloid precursor protein by cathepsin D.

The events leading to the formation of beta-amyloid (betaA4) from its precursor (betaAPP) involve proteolytic cleavages that produce the amino and carboxyl termini of betaA4. The enzyme activities responsible for these cleavages have been termed beta- and gamma-secretase, respectively, although these protease(s) have not been identified. Since betaA4 is known to possess heterogeneity at both the amino and carboxyl termini, beta- and gamma-secretases may actually be a collection of proteolytic activities or perhaps a single proteolytic enzyme with broad amino acid specificity. We investigated the role of cathepsin D in the processing of betaAPP since this enzyme has been widely proposed as a gamma-secretase candidate. Treatment of a synthetic peptide that spans the gamma-secretase site of betaAPP with human cathepsin D resulted in the cleavage of this substrate at Ala42-Thr43. A sensitive liquid chromatography/mass spectrometry technique was also developed to further investigate the ability of cathepsin D to process longer recombinant betaAPP substrates (156 and 100 amino acids of betaAPP carboxyl terminus) in vitro. The precise cathepsin D cleavage sites within these recombinant betaAPP substrates were identified using this technique. Both recombinant substrates were cleaved at the following sites: Leu49-Val50, Asp68-Ala69, Phe93-Phe94. No cleavages were observed at putative gamma-secretase sites: Val40-Ile41 or Ala42-Thr43, suggesting that cathepsin D is not gamma-secretase as defined by these betaA4 termini. Under conditions where the betaAPP156 substrate was first denatured prior to cathepsin D digestion, two additional cleavage sites near the amino terminus of betaA4, Glu-3-Val-2 and Glu3-Phe4, were observed, indicating that cathepsin D cleavage of betaAPP is influenced by the structural integrity of the substrate. Taken together, these results indicate that in vitro, cathepsin D is unlikely to function as gamma-secretase; however, the ability of this enzyme to efficiently cleave betaAPP substrates at nonamyloidogenic sites within the molecule may reflect a role in betaAPP catabolism.

Characterization of the tryptic map of recombinant DNA derived tissue plasminogen activator by high-performance liquid chromatography-electrospray ionization mass spectrometry.

A detailed tryptic map is presented for recombinant human tissue plasminogen activator (rt-PA). Electrospray ionization mass spectrometry is utilized as an on-line HPLC detector for tryptic mapping of this glycoprotein. The additional dimension provided by mass spectrometry gives considerably more detail about the complex tryptic map and significantly enhances the high-resolution chromatographic separation by distinguishing by mass any coeluting components. Through this improvement, the proline isomers of a tryptic peptide were observed eluting over a broad range of retention times. The glycopeptides of rt-PA are observed as well as any corresponding nonglycosylated peptides. In addition, the carbohydrate heterogeneity is readily observed, allowing analysis of the carbohydrate composition. The characteristic diagonal patterns formed by glycopeptides in a contour plot of the data allow rapid recognition of the glycopeptides.