PubMed Health⌕ Search

Biomedical subjects

J J Gorman

Publications and source records attributed to J J Gorman.

At least 19 recordsLinked to original sources
PubMed2004

Immunoproteomics: Mass spectrometry-based methods to study the targets of the immune response.

The mammalian immune system has evolved to display fragments of protein antigens derived from microbial pathogens to immune effector cells. These fragments are typically peptides liberated from the intact antigens through distinct proteolytic mechanisms that are subsequently transported to the cell surface bound to chaperone-like receptors known as major histocompatibility complex (MHC) molecules. These complexes are then scrutinized by effector T cells that express clonally distributed T cell receptors with specificity for specific MHC-peptide complexes. In normal uninfected cells, this process of antigen processing and presentation occurs continuously, with the resultant array of self-antigen-derived peptides displayed on the surface of these cells. Changes in this peptide landscape of cells act to alert immune effector cells to changes in the intracellular environment that may be associated with infection, malignant transformation, or other abnormal cellular processes, resulting in a cascade of events that result in their elimination. Because peptides play such a crucial role in informing the immune system of infection with viral or microbial pathogens and the transformation of cells in malignancy, the tools of proteomics, in particular mass spectrometry, are ideally suited to study these immune responses at a molecular level. Here we review recent advances in the studies of immune responses that have utilized mass spectrometry and associated technologies, with specific examples from collaboration between our laboratories.

Antigen Presentation↗
PubMed2001

Cloning, expression, and crystallization of the fusion protein of Newcastle disease virus.

We have recently reported the X-ray crystal structure of a fragment of the fusion protein (F) of Newcastle disease virus (NDV). This work describes the methodology involved in the production and crystallization of that protein in recombinant form. The full-length cDNA of NDV-F was cloned and the ectodomain expressed in both CHO-K1 and Lec-3.2.8.1 cells. The recombinant protein, secreted as a single-chain polypeptide F0', was purified using a c-myc antibody affinity column followed by gel filtration chromatography. Electron microscopic imaging showed the F0' product to consist of unaggregated club-shaped particles. Trypsin treatment of F0' could be used to produce disulfide-linked F2 and F1' chains. However, imaging revealed extensive rosette-like aggregation of the trypsin-treated material, indicative of a conformational change. Only the non-trypsin-treated product was thus suitable for crystallization and two crystal forms were obtained, diffracting to ca. 3.5 and 4.0 A, respectively. Both crystal forms were used in the structure determination.

Amino Acid Sequence↗
PubMed2001

Antiviral activity and structural characteristics of the nonglycosylated central subdomain of human respiratory syncytial virus attachment (G) glycoprotein.

Segments of the cystine noose-containing nonglycosylated central subdomain, residues 149-197, of the attachment (G) glycoprotein of human respiratory syncytial virus (HRSV) have been assessed for impact on the cytopathic effect (CPE) of respiratory syncytial virus (RSV). Nalpha-acetyl residues 149-197-amide (G149-197), G149-189, and G149-177 of the A2 strain of HRSV protected 50% of human epithelial HEp-2 cells from the CPE of the A2 strain at concentrations (IC(50)) between 5 and 80 microm. Cystine noose-containing peptides G171-197 and G173-197 did not inhibit the CPE even at concentrations above 150 microm. Systematic C- and N-terminal truncations from G149-189 and G149-177 and alanine substitutions within G154-177 demonstrated that residues 166-170 (EVFNF), within a sequence that is conserved in HRSV strains, were critical for inhibition. Concordantly, G154-177 of bovine RSV and of an antibody escape mutant of HRSV with residues 166-170 of QTLPY and EVSNP, respectively, were not inhibitory. Surprisingly, a variant of G154-177 with an E166A substitution had an IC(50) of 750 nm. NMR analysis demonstrated that G149-177 adopted a well-defined conformation in solution, clustered around F168 and F170. G154-170, particularly EVFNF, may be important in binding of RSV to host cells. These findings constitute a promising platform for the development of antiviral agents for RSV.

Alanine↗
PubMed2001

The structure of the fusion glycoprotein of Newcastle disease virus suggests a novel paradigm for the molecular mechanism of membrane fusion.

BACKGROUND: Membrane fusion within the Paramyxoviridae family of viruses is mediated by a surface glycoprotein termed the "F", or fusion, protein. Membrane fusion is assumed to involve a series of structural transitions of F from a metastable (prefusion) state to a highly stable (postfusion) state. No detail is available at the atomic level regarding the metastable form of these proteins or regarding the transitions accompanying fusion. RESULTS: The three-dimensional structure of the fusion protein of Newcastle disease virus (NDV-F) has been determined. The trimeric NDV-F molecule is organized into head, neck, and stalk regions. The head is comprised of a highly twisted beta domain and an additional immunoglobulin-like beta domain. The neck is formed by the C-terminal extension of the heptad repeat region HR-A, capped by a four-helical bundle. The C terminus of HR-A is encased by a further helix HR-C and a 4-stranded beta sheet. The stalk is formed by the remaining visible portion of HR-A and by polypeptide immediately N-terminal to the C-terminal heptad repeat region HR-B. An axial channel extends through the head and neck and is fenestrated by three large radial channels located approximately at the head-neck interface. CONCLUSION: We propose that prior to fusion activation, the hydrophobic fusion peptides in NDV-F are sequestered within the radial channels within the head, with the central HR-A coiled coil being only partly formed. Fusion activation then involves, inter alia, the assembly of a complete HR-A coiled coil, with the fusion peptides and transmembrane anchors being brought into close proximity. The structure of NDV-F is fundamentally different than that of influenza virus hemagglutinin, in that the central coiled coil is in the opposite orientation with respect to the viral membrane.

Amino Acid Sequence↗
PubMed2001

The use of post-source decay in matrix-assisted laser desorption/ionisation mass spectrometry to delineate T cell determinants.

The identification of naturally processed peptides presented by molecules of the major histocompatibility complex (MHC) has progressed significantly over the past decade. The elution of peptides from immunoaffinity purified complexes of MHC class I or class II molecules has provided highly specific biochemical information regarding the nature of endogenous peptides capable of binding to and being presented by particular MHC alleles. Whilst Edman chemistry is sufficient for the identification of abundant or homogeneous immunodominant peptides contained in samples of fractionated peptides, mass spectrometry has proved more powerful for sequencing less abundant species present in the typically heterogeneous fractions of eluted peptides. This review focuses on the characterisation of T cell determinants by matrix-assisted laser desorption/ionisation (MALDI)-time-of-flight (TOF) mass spectrometry (MS). We demonstrate, with specific examples, the utility of post-source decay in MALDI-TOF MS for the characterisation of the amino acid sequences of both native and modified T cell determinants. The potential advantages and pitfalls of this technique relative to the more commonly used forms of tandem mass spectrometry in electrospray and ion spray modes of ionisation as well as hybrid quadrupole-quadrupole-TOF instruments are discussed. We highlight the complementarity between these techniques and discuss the advantages in the combined use of both MALDI- and electrospray-based instrumentation in epitope identification strategies.

Animals↗
PubMed2001

Quantitative and qualitative influences of tapasin on the class I peptide repertoire.

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and beta(2)-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.

Antigen Presentation↗
PubMed2001

Identification of disulfide-linked peptides by isotope profiles produced by peptic digestion of proteins in 50% (18)O water.

Determination of the disulfide-bond arrangement of a protein by characterization of disulfide-linked peptides in proteolytic digests may be complicated by resistance of the protein to specific proteases, disulfide interchange, and/or production of extremely complex mixtures by less specific proteolysis. In this study, mass spectrometry has been used to show that incorporation of (18)O into peptides during peptic digestion of disulfide-linked proteins in 50% (18)O water resulted in isotope patterns and increases in average masses that facilitated identification and characterization of disulfide-linked peptides even in complex mixtures, without the need for reference digests in 100% (16)O water. This is exemplified by analysis of peptic digests of model proteins lysozyme and ribonuclease A (RNaseA) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). Distinct isotope profiles were evident when two peptide chains were linked by disulfide bonds, provided one of the chains did not contain the C terminus of the protein. This latter class of peptide, and single-chain peptides containing an intrachain disulfide bond, could be identified and characterized by mass shifts produced by reduction. Reduction also served to confirm other assignments. Isotope profiling of peptic digests showed that disulfide-linked peptides were often enriched in the high molecular weight fractions produced by size exclusion chromatography (SEC) of the digests. Applicability of these procedures to analysis of a more complex disulfide-bond arrangement was shown with the hemagglutinin/neuraminidase of Newcastle disease virus.

Amino Acid Sequence↗
PubMed2000

Tapasin-mediated retention and optimization of peptide ligands during the assembly of class I molecules.

The murine class I H-2Kb molecule achieves high level surface expression in tapasin-deficient 721.220 human cells. Compared with their behavior in wild-type cells, Kb molecules expressed on 721.220 cells are more receptive to exogenous peptide, undergo more rapid surface decay, and fail to form macromolecular peptide loading complexes. As a result, they are rapidly transported to the cell surface, reflecting a failure of endoplasmic reticulum retention mechanisms in the absence of loading complex formation. Despite the failure of Kb molecules to colocalize to the TAP and their rapid egress to the cell surface, Kb is still capable of presenting TAP-dependent peptides in the absence of tapasin. Furthermore, pool sequencing of peptides eluted from these molecules revealed strict conservation of their canonical H-2Kb-binding motif. There was a reduction in the total recovery of peptides associated with Kb molecules purified from the surface of tapasin-deficient cells. Comparison of the peptides bound to Kb in the presence and absence of tapasin revealed considerable overlap in peptide repertoire. These results indicate that in the absence of an interaction with tapasin, Kb molecules fail to assemble with calreticulin and TAP, yet they are still capable of acquiring a diverse array of peptides. However, a significant proportion of these peptides appear to be suboptimal, resulting in reduced cell surface stability of Kb complexes. Taken together, the findings indicate that tapasin plays an essential role in the formation of the class I loading complex, which retains class I heterodimers in the endoplasmic reticulum until optimal ligand selection is completed.

Animals↗
PubMed2000

Determination of the disulfide bond arrangement of Newcastle disease virus hemagglutinin neuraminidase. Correlation with a beta-sheet propeller structural fold predicted for paramyxoviridae attachment proteins.

Disulfide bonds stabilize the structure and functions of the hemagglutinin neuraminidase attachment glycoprotein (HN) of Newcastle disease virus. Until this study, the disulfide linkages of this HN and structurally similar attachment proteins of other members of the paramyxoviridae family were undefined. To define these linkages, disulfide-linked peptides were produced by peptic digestion of purified HN ectodomains of the Queensland strain of Newcastle disease virus, isolated by reverse phase high performance liquid chromatography, and analyzed by mass spectrometry. Analysis of peptides containing a single disulfide bond revealed Cys(531)-Cys(542) and Cys(172)-Cys(196) linkages and that HN ectodomains dimerize via Cys(123). Another peptide, with a chain containing Cys(186) linked to a chain containing Cys(238), Cys(247), and Cys(251), was cleaved at Met(249) with cyanogen bromide. Subsequent tandem mass spectrometry established Cys(186)-Cys(247) and Cys(238)-Cys(251) linkages. A glycopeptide with a chain containing Cys(344) linked to a chain containing Cys(455), Cys(461), and Cys(465) was treated sequentially with peptide-N-glycosidase F and trypsin. Further treatment of this peptide by one round of manual Edman degradation or tandem mass spectrometry established Cys(344)-Cys(461) and Cys(455)-Cys(465) linkages. These data, establishing the disulfide linkages of all thirteen cysteines of this protein, are consistent with published predictions that the paramyxoviridae HN forms a beta-propeller structural fold.

Amidohydrolases↗
PubMed1999

Pathotyping isolates of Newcastle disease virus using antipeptide antibodies to pathotype-specific regions of their fusion and hemagglutinin-neuraminidase proteins.

Antipeptide antibodies have been evaluated for their abilities to predict the characteristics of the cleavage motifs of the fusion protein precursors (F0) of 25 isolates of Newcastle disease virus (NDV) with a range of virulences, grouped into 12 sets according to their monoclonal antibody reactivities. A Western blot format was used to show that antisera to synthetic peptides representing sequences at the C-termini of the F2-polypeptides of defined pathotypes of NDV usually distinguish between pathotypes on the basis of their Fo cleavage sequences. However, exceptions were found with three groups of virulent isolates. Protein sequencing and mass spectral analysis of the F2-polypeptide of isolate Texas GB from one of these groups, identified an anomalous cleavage/activation process which removed the amino acids required for recognition by the antisera. This probably also explained the lack of reactivity of the Roakin isolate and low reactivity of the Komarov isolate from this group. The other exceptions involved isolates in groups with cleavage region variations from the usual motif of virulent isolates or isolates with undefined cleavage motifs. Antipeptide antisera were also raised to sections of the 45 residue C-terminal extension the hemagglutinin-neuraminidase precursor (HN0) encoded by the genes of some avirulent isolates. Western blot analysis showed that positive reactions with antibodies to peptides based on sequences between residues 577 and 613 of the HN0 was evidence for the presence of an avirulent isolate but did not exclude the presence of other pathotypes. Antisera designed to target residues 569-577 detected HN0 extensions of 6 residues on isolates known to encode such extensions. These antisera also enabled differentiation of isolates with HN0 extensions of 6 residues from those with no extension, however, it was not possible to determine the virulence of isolates based on reaction with these antisera.

Amino Acid Sequence↗
PubMed1998

Avoidance of self-reactivity results in skewed CTL responses to rare components of synthetic immunogens.

In studying the CTL recognition of peptide determinants derived from the nuclear Ag La (SS-B), we observed significant skewing of the response toward rare components present within the immunogen. Thus, priming of naive mouse lymphocytes in vitro with a synthetic H-2Kb-binding peptide comprising human La (hLa) residues 51-58 resulted in class I-restricted cytotoxic T cells that failed to recognize naturally presented hLa 51-58 peptide. Instead, the majority of T hybrids recognized a low abundance (< or = 1%) contaminant present at picomolar concentrations in the original synthesis and identified as a peptide adduct containing N,4-t-butyl asparagine at position 6 of the hLa 51-58 sequence. The preferred T cell recognition of the butyl adduct was not due to increased affinity of this peptide for the H-2Kb molecule or to the antagonism of CTL recognizing the unmodified determinant. Rather, the bias in the immune response appeared to be the result of partial self-tolerance to the homologous mouse La 51-58 determinant, which differs from its human counterpart by only a single amino acid at position 1 (T-->I). Accordingly, the CTL response appeared to be focused on "non-self" ligands present within the synthesis, even though they were present at very low concentrations. These observations have significant implications for the use of synthetic peptide vaccines, especially those designed to manipulate responses to self peptides such as tumor Ags in which self-tolerance may result in unexpected reactivity.

Amino Acid Substitution↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1997

The disulfide bonds in the C-terminal domains of the human insulin receptor ectodomain.

The human insulin receptor is a homodimer consisting of two monomers linked by disulfide bonds. Each monomer comprises an alpha-chain that is entirely extracellular and a beta-chain that spans the cell membrane. The alpha-chain has a total of 37 cysteine residues, most of which form intrachain disulfide bonds, whereas the beta-chain contains 10 cysteine residues, four of which are in the extracellular region. There are two classes of disulfide bonds in the insulin receptor, those that can be reduced under mild reducing conditions to give alpha-beta monomers (class I) and those that require stronger reducing conditions (class II). The number of class I disulfides is small and includes the alpha-alpha dimer bond Cys524. In this report we describe the use of cyanogen bromide and protease digestion of the exon 11 plus form of the receptor ectodomain to identify disulfide linkages between the beta-chain residues Cys798 and Cys807 and between the alpha-chain Cys647 and the beta-chain Cys872. The latter bond is the sole alpha-beta link in the molecule and implies a side-by-side alignment of the two fibronectin III domains of the receptor. Also presented is evidence for additional alpha-alpha dimer bond(s) involving at least one of the cysteine residues of the triplet at positions 682, 683, and 685. Evidence is also presented to show that Cys884 exists as a buried thiol in the soluble ectodomain.

Amino Acid Sequence↗
PubMed1997

Oligosaccharide characterization and quantitation using 1-phenyl-3-methyl-5-pyrazolone derivatization and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of monosaccharides, maltooligosaccharides, and oligosaccharides enzymatically released from asparagine-linked sites in ribonuclease B and fetuin have been investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Use of the matrix 2,6-dihydroxyacetophenone containing diammonium hydrogen citrate (DHAP/DAHC) resulted in predominance of protonated over sodiated pseudomolecular ions of PMP-derivatized oligosaccharides. By comparison, the matrices alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid resulted in predominantly sodiated pseudomolecular ions. In addition, tendencies for fragmentation of PMP-oligosaccharide derivatives were significantly lower with DHAP/DAHC which enabled meaningful data to be obtained in reflector mode, even for samples with high excipient levels. The relative magnitude of the ion signals for PMP-derivatized maltooligosaccharides and ribonuclease B oligosaccharides correlated well with the oligomer distribution apparent by HPLC. PMP-maltohexose was used as an internal standard to quantitate PMP-oligosaccharides from ribonuclease B and asialofetuin in crude derivatization mixtures. A linear relationship was observed between the ratio of the intensities of pseudomolecualr ions and the amount of glycoprotein derivatized. The limit of detection for the major oligosaccharide of each protein was reached with ca. 3 micrograms of glycoprotein but may be further enhanced by optimization of sample handling. PMP derivatives of sialylated fetuin oligosaccharides were readily detected as protonated pseudomolecular ions by linear mode analyses. By comparison, reflector mode analyses revealed substantially reduced magnitudes of protonated pseudomolecular ions and considerable post-source fragmentation of sialic acid residues. The PMP derivatives of fetuin oligosaccharides were also amenable to exoglycosidase treatment as shown by the mass shifts found upon treatment with sialidase.

Antipyrine↗
PubMed1997

Determination of the disulfide bond arrangement of human respiratory syncytial virus attachment (G) protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The attachment protein or G protein of the A2 strain of human respiratory syncytial virus (RSV) was digested with trypsin and the resultant peptides separated by reverse-phase high-performance liquid chromatography (HPLC). One tryptic peptide produced a mass by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) corresponding to residues 152-187 with the four Cys residues of the ectodomain (residues 173, 176, 182, and 186) in disulfide linkage and absence of glycosylation. Sub-digestion of this tryptic peptide with pepsin and thermolysin produced peptides consistent with disulfide bonds between Cys173 and Cys186 and between Cys176 and Cys182. Analysis of ions produced by post-source decay of a peptic peptide during MALDI-TOF-MS revealed fragmentation of peptide bonds with minimal fission of an inter-chain disulfide bond. Ions produced by this unprecedented MALDI-induced post-source fragmentation corroborated the existence of the disulfide arrangement deduced from mass analysis of proteolysis products. These findings indicate that the ectodomain of the G protein has a non-glycosylated subdomain containing a "cystine noose."

Amino Acid Sequence↗
PubMed1996

Use of 2,6-dihydroxyacetophenone for analysis of fragile peptides, disulphide bonding and small proteins by matrix-assisted laser desorption/ionization.

Several peptides were shown to undergo fragmentation during matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to a degree which complicated their analysis using alpha-cyano-4-hydroxycinnamic acid (CHCA) as a matrix, even at threshold laser irradiance. These peptides included synthetic peptides, peptides isolated from viral proteins and a phosphopeptide from beta-casein (residues 33-48). The excessive fragmentation occurred usually as a post-source phenomenon; however, in-source fragmentation was also observed. The combined effects of in-source and post-source fragmentation of one peptide studied led to a failure to observe the protonated molecule of this peptide in reflector mode analysis. The phosphopeptide studied exhibited a high degree of beta-elimination of phosphate. It was demonstrated that the fragility exhibited by these peptides in CHCA, including beta-elimination of phosphate from serine, was not evident with a matrix comprising 2,6-dihydroxyacetophenone (DHAP) and di-ammonium hydrogen citrate (DAHC). The DHAP/DAHC matrix was also adapted for direct analysis of peptides from an acidic reducing milieu containing tris(2-carboxyethyl)phosphine. The molecular weight of equine cytochrome c was determined with a relatively high degree of accuracy (experimental M(r) = 12360.2 +/- 1.4 Da compared to the theoretical M(r) = 12360.09 Da) using DHAP/DAHC as a matrix for reflector mode analysis.

Acetophenones↗
PubMed1993

Isolation of carboxyl-termini and blocked amino-termini of viral proteins by high-performance cation-exchange chromatography.

The strong cation-exchanger, PolySulfoethyl Aspartamide, has been assessed as a medium for isolation of carboxyl-terminal and blocked amino-terminal peptides from tryptic digests of small quantities of viral proteins. Peptides with a single positive charge, the blocked amino-terminal peptides of ovalbumin and the Newcastle disease virus (NDV) matrix protein and carboxyl-terminal peptides of ovalbumin and the NDV nucleocapsid protein, eluted in early ion-exchange fractions and were readily isolated in homogeneous form by subsequent reversed-phase HPLC. Some early ion-exchange fractions also contained singly charged peptides derived by "chymotryptic-like" cleavage, whilst other peptides eluted in these fractions due to their highly acidic character. Terminal sequences with additional basic residues were isolated from later eluting ion-exchange fractions. Peptides with this property included the blocked amino-terminus of the NDV nucleocapsid protein and a portion of the carboxyl-terminus of the NDV matrix protein. Hitherto undescribed polymorphism in the amino-terminal region of ovalbumin was revealed in this study. Truncated peptides from the carboxyl-terminus of the NDV matrix protein were also detected. The presence of these peptides could be a reflection of carboxyl-terminal processing of the matrix protein. The strategy described herein should be of general utility for selective microisolation of carboxyl-terminal peptides and blocked amino-terminal peptides from tryptic digests of proteins.

Amino Acids↗