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We have identified the residue in Cellulomonas fimi exoglycanase modified by N-bromoacetyl cellobiosylamine as Glu 127 using a new combination of experimental approaches. The enzyme was quantitatively inhibited with the affinity label N-bromoacetyl cellobiosylamine and cleaved with pepsin. The N-acetyl cellobiosylamine-modified peptide was identified by comparative peptide mapping of the digests derived from labeled and unlabeled proteins by reverse-phase high-performance liquid chromatography connected online to an electrospray ionization mass spectrometer. The modified residue in the labeled peptide was determined by using a novel protein sequencing chemistry which is based on monitoring the amino acid derivatives released by stepwise peptide degradation using electrospray ionization mass spectrometry. Tandem mass spectrometry was used for further structural characterization of the cleaved residue. We show that the residue modified by N-bromoacetyl cellobiosylamine is Glu 127. This residue has been identified previously as the acid-base catalyst by using a combination of mutagenic and kinetic analyses. Our results therefore demonstrate the usefulness of this type of affinity label in identifying important catalytic residues in glycosidases and suggest that this new experimental approach can be applied generally to any labeled protein in which the mass of the label is known and thus represents an alternative approach to the current methods used to identify labeled residues within proteins.

We constructed a resin-bound peptide library in which the peptide was designed to take an α-helix structure, and p-boronobenzoic acid was conjugated on the e-amino group of the Lys side-chain place...

We report a general mass spectrometric approach for the rapid identification and characterization of proteins isolated by preparative two-dimensional polyacrylamide gel electrophoresis. This method possesses the inherent power to detect and structurally characterize covalent modifications. Absolute sensitivities of matrix-assisted laser desorption ionization and high-energy collision-induced dissociation tandem mass spectrometry are exploited to determine the mass and sequence of subpicomole sample quantities of tryptic peptides. These data permit mass matching and sequence homology searching of computerized peptide mass and protein sequence data bases for known proteins and design of oligonucleotide probes for cloning unknown proteins. We have identified 11 proteins in lysates of human A375 melanoma cells, including: alpha-enolase, cytokeratin, stathmin, protein disulfide isomerase, tropomyosin, Cu/Zn superoxide dismutase, nucleoside diphosphate kinase A, galaptin, and triosephosphate isomerase. We have characterized several posttranslational modifications and chemical modifications that may result from electrophoresis or subsequent sample processing steps. Detection of comigrating and covalently modified proteins illustrates the necessity of peptide sequencing and the advantages of tandem mass spectrometry to reliably and unambiguously establish the identity of each protein. This technology paves the way for studies of cell-type dependent gene expression and studies of large suites of cellular proteins with unprecedented speed and rigor to provide information complementary to the ongoing Human Genome Project.

When murine interleukin-6 is overexpressed in Escherichia coli, a small population of molecules exhibits a novel C-terminal modification. Peptide mapping, electrospray ionization-mass spectrometry, and automated N- and C-terminal sequencing identified a peptide ("tag" peptide), -Ala-Ala-Asn-Asp-Glu-Asn-Tyr-Ala-Leu-Ala-Ala-COOH, encoded by a small metabolically stable RNA of E. coli (10Sa RNA) attached to truncated C termini of the recombinant protein. A mutant strain of E. coli in which the chromosomal 10Sa RNA gene (ssrA) is disrupted does not produce this C-terminal modification, confirming that the tag peptide originates from the ssrA gene.

Tandem mass spectrometry is a popular tool for the identification of peptide sequences. In this paper, we present a method for a rapid generation of short peptide sequences via tandem mass spectrometry based on a graph search method. The approach takes advantage of several pairs of peaks that have high intensities. We proposed a Pair Peak value Set (PPS) and used the pair peak values of highest intensities as the root of a tree. The other nodes are viewed as the reference nodes to search the most promising path. We aimed to determine the peptide sequences for MS/MS spectra that have low signal-to-noise ratios. Our experiment on 2420 experimental MS/MS spectra with two PTMs shows that our algorithm achieves better accuracy than the PepNovo approach with higher efficiency.

We demonstrate a new approach to the identification of mass spectrometrically fragmented peptides. A fragmentation spectrum usually contains a short, easily identifiable series of sequence ions, which yields a partial sequence. This partial sequence divides the peptide into three parts-regions 1, 2, and 3-characterized by the added mass m1 of region 1, the partial sequence of region 2, and the added mass m3 of region 3. We call the construct, m1 partial sequence m3, a "peptide sequence tag" and show that it is a highly specific identifier of the peptide. An algorithm developed here that uses the sequence tag to find the peptide in a sequence database is up to 1 million-fold more discriminating than the partial sequence information alone. Peptides can be identified even in the presence of an unknown posttranslational modification or an amino acid substitution between an entry in the sequence database and the measured peptide. These concepts are demonstrated with model and practical examples of electrospray mass spectrometry/mass spectrometry of tryptic peptides. Just two to three amino acid residues derived by fragmentation are enough to identify these peptides. In peptide mapping applications, even less information is necessary.

The base sequence of synthetic peptide nucleic acids (PNAs), novel antisense agents, was analyzed by positive-ion fast-atom bombardment tandem mass spectrometry (FAB-MS/MS). Upon high-energy collisional activation decomposition, PNA oligomers provided apparent MS/MS product ions resulting from specific cleavage along the PNA backbone.

GAP-43 isolated from calf brain was analyzed by the electrospray mass spectrometry. The mass spectrum of the intact protein showed two species with a mass difference of 80 Da, suggesting that the isolated GAP-43 contains phosphorylated species. To establish the in vivo phosphorylation sites, the protein was digested with trypsin, and analyzed by the liquid chromatography/mass spectrometry technique, in which a capillary reversed-phase chromatography column was connected on line to an electrospray mass spectrometer. Two pairs of peptides with a mass difference of 80 Da were observed. From the tandem mass spectrometry, two novel phosphorylation sites (Thr-87 and Ser-152) were identified. The novel phosphorylation sites contain proline immediately after the phosphorylated serines. No phosphorylated peptide was detected corresponding to the protein kinase C or casein kinase II phosphorylation sites. A peptide corresponding to the acetylated N-terminal peptide was also identified. The mass of the peptide suggests that the 2 cysteinyl residues are not palmitoylated but form a disulfide bridge.

A new strategy has been developed for the determination of the amino acid sequence of apamin, a two disulfide-bond neurotoxic oligopeptide. Relative molecular mass up to about 2000 was determined using electrospray mass spectrometry. This technique shows that three basic amino acid residues are present in apamin. A hydrophobic tryptic digest fragment was sequenced using fast-atom bombardment combined with tandem mass spectrometry. But the full sequence has been directly observed using a tandem mass spectrometer: the daughter-ion mass spectrum of protonated molecules of dithiothreitol-reduced apamin gives the sequence.

Publisher Summary This chapter describes the optimization and application of combined Edman-chemical and laser-desorption mass spectrometric approaches to micro peptide sequencing. Transactions among membrane-bound compartments in the cytoplasm of eukaryotic cells are generally executed by transport vesicles that bud from one membrane and fuse selectively with another. Peptide fractions T11 and T12 yielded two and three peak m / z values, respectively, when analyzed by MALDI-MS, and the results indicated overlapping peptide species to be present in both fractions, likely because of cross-contamination after high-performance liquid chromatography. Unfortunately, although the sequencer-instrument was judged to be in perfect working condition, only a snippet of sequence was obtained for T12 and T11 failed to give any sequence at all. One of the three m/z values for T12 was in excellent agreement with the value for the predicted tryptic peptide containing the limited sequence, so confirming the identity of the entire peptide.

Publisher Summary This chapter describes the identification of 2D gel proteins at the femtomole level by molecular mass searching of peptide fragments in a protein sequence database. In a study described in the chapter, single 2D gel spots were excised from the PVDF membrane and wetted with 1 μl of methanol. A single match with Escherichia coli uridine phosphorylase was obtained from more than 100,000 proteins in the database. The identity of this protein was confirmed by N-terminal analysis of spot 12 from an identical gel. The initial yield was used to estimate the amount of protein in spot 12 and the amount used for matrix-assisted laser desorption/ionization analysis. The partial fragment option, which allows matches with partially digested fragments, was used. This allowed a match with mass 2109.3, which corresponds to a peptide that contains an undipped arginine residue. When a search was run without the partial option, the program obtained the same results except mass 2109.3, which corresponded to the partially cleaved peptide, was not found. The use of an enzyme blank can eliminate masses derived from autolysis of the protease that can occur during digestion of 2D gel spots.

As we have seen in Chapter 1, what uniquely defines a specific protein is its amino acid sequence, or primary structure. The methodology for determining the sequential arrangement of amino acids in a protein or peptide has been in place for some time. The first protein for which a full amino acid sequence was determined was the peptide hormone insulin by Frederick Sanger in 1953. At the time, this work was considered a major breakthrough, since it proved that proteins have uniquely defined structures. Sanger received the Nobel Prize for this work in 1958. While much of the chemistry involved in amino acid sequencing is now automated, the determination of protein sequences remains a tedious and technically demanding effort that is best left to laboratories that specialize in these methods. Nevertheless, the generalist can gain insight into the structure of target proteins by a number of methods that provide indirect information of amino acid composition and arrangement. Peptide mapping and amino acid analysis are often combined to provide this type of information. In this chapter we shall describe the methods commonly employed for peptide mapping and amino acid analysis of proteins. As we shall see, these methods are readily accessible to most protein scientists. At the end of the chapter, we shall briefly discuss the basis for protein sequence analysis. Again, the actual determination of protein sequences is largely the realm of experts, but it is worthwhile reviewing the chemistry involved in sequence analysis, since this is such an important part of modern protein science.

A computer searching algorithm has been used to identify protein sequences in the Protein Information Resource (PIR) database with peptide mass information (mass map) obtained from proteolytic digests of proteins analyzed by microcapillary high-performance liquid chromatography electrospray ionization mass spectrometry. A theoretical analysis of the cytochrome c family demonstrates the ability to identify protein sequences in the PIR database with a high degree of accuracy using a set of six predicted tryptic peptide masses. This method was also applied to experimentally determined peptide masses for a small GTP-binding protein, a protein from pig uterus, the human sex steroid binding protein, and a thermostable DNA polymerase. The results demonstrate that a set of observed masses which is less than 50% of the total number of predicted masses can be used to identify a protein sequence in the database. For the analysis presented in this paper, a mass matching tolerance of 1 amu is used. Under these conditions, mass maps created by fast atom bombardment mass spectrometry and matrix-assisted laser desorption time-of-flight would also be applicable. In cases where multiple matches are observed or verification of the protein identification is needed, tandem mass spectrometry sequencing can be used to establish sequence similarity.

The application of pattern recognition to sequence elucidation from fast atom bombardment (FAB), collisionally activated dissociation (CAD), and tandem mass spectrometric data of peptides was investigated. Learning machine techniques for pattern recognition were applied to detect C-terminal series amino acid sequences up to the pentapeptide Try-Gly-Gly-Phe-Leu (YGGFL). The approach conditions the data by building upon known fragmentation pathways of peptides in FAB/CAD-related analysis. The intensities of critical sequence ion peaks are used to describe each pattern in the training set. A well-defined training set is then made use of to classify unknown species. The FORTRAN-77 program is adapted from one first developed by P.C. Jurs. It requires only the input of the critical peak intensities and the length of the unit to be tested. The method has potential in applications to larger peptides provided a database for the training set can be constructed.

The utility of hydrogen-deuterium exchange for sequencing peptides by mass spectrometry is demonstrated. The number of exchangeable hydrogens in a peptide is readily obtained by electrospray analysis of the peptide dissolved in deuterated solvents. This information can be used, in conjunction with published computer algorithms for interpreting peptide mass spectra, to reduce significantly the number of candidate sequences that fit the experimental data. This information, when combined with fragment-ion information in the mass spectrum, greatly increases the reliability of sequence determination.

The combination of collision-induced dissociation (CID) and linked-scan analysis was used for analysing the sequence ions from the precursor ion of a peptide, which had been labelled with 18O at its carboxyl terminus (C-terminus) using 40 atom % H2 18O. The CID and linked-scan mass spectrum of the labelled peptide gave two series of sequence-ion signals: the one, originating from the C-terminus of the labelled peptide, showed a doublet signal due to the part-incorporation of 18O into the carboxyl group at the C-terminus, while the other, originating from the amino terminus (N-terminus), has the natural isotopic ion distribution. From the distribution of the isotopic ions in a single CID spectrum, the sequence ions containing the C-terminus could be readily differentiated from those containing the N-terminus, allowing the facile assignment of sequence ions to the amino-acid sequence of a peptide by CID and linked-scan analysis. This method was successfully applied to determination of the amino-acid sequence of the light-chain of mouse anti-porphyrin monoclonal antibody.

A method has been developed for the determination of the amino-acid sequence of a cyclic peptide containing cystine. It is based on the reduction of the peptide in a reductive matrix prior to ionization by fast-atom bombardment. The amino-acid sequence of the resulting linear peptide is then determined by tandem mass spectrometry from the spectrum produced by the collision-induced decomposition of the [M + H]+ ion of the peptide.

A new method for characterizing O-linked glycopeptides without chemical degradation is presented. Collision-induced dissociation (CID) analysis of intact O-linked glycopeptides containing mono- and disaccharides was performed. For glycopeptides containing one hexose unit, both the peptide sequence and the site of attachment of the sugar moiety were obtained from a single high-energy CID spectrum. However, in a glycopeptide bearing multiple sugar residues per site, the CID spectrum was dominated by fragments resulting from cleavages of the carbohydrate substituents and the gas-phase deglycosylated peptide, thus obviating the concomitant observation of peptide sequence ions. Hence, information on the structures of the carbohydrate substituents was obtained, but not on the sites of attachment of these residues to the peptide. Subsequent CID analysis of the gas-phase deglycosylated peptide ion can be used to obtain the sequence of the peptide backbone from the same sample. This method holds promise for simultaneously determining the carbohydrate structure and the peptide sequence of intact O-linked glycopeptides without chemical degradation.

cDNA transcripts encoding rat monitor peptide (MP) have been cloned from a lambda-ZAP-II phage library using minimal specific amino acid sequence (six residues), the polymerase chain reaction (PCR), and multivalent PCR probes to distinguish MP transcripts from those that encode a closely related peptide, pancreatic secretory trypsin inhibitor. DNA sequence analysis of 3 cDNA transcripts, MP1-3, revealed the complete amino acid sequence of the prepeptide (79 residues) including an 18-residue hydrophobic signal sequence at the NH2 terminus. Sequence divergence in both coding and 3' noncoding regions indicates a potential exon-exon junction with alternative splicing, which results in a truncated peptide with Arg 58 at the COOH terminus as well as alternative selection of poly(A) signals, respectively. The 5' nontranslated region of MP1 mRNA (282 nucleotides (nt] contains four upstream ATGs. Conserved structure between MP and anionic trypsinogen mRNAs within 9 nt immediately upstream of the AUG initiation codon may be involved in coupling the expression of MP with anionic trypsinogen, a condition which appears to be required to monitor the intake of dietary protein in the rat.

The use of electrospray tandem mass spectrometry is demonstrated for the characterisation of the polypeptide chains of normal human haemoglobin and a number of β-chain variants. Rapid screening of aberrant haemoglobins is achieved by the analysis of diluted blood without need for pretreatment or sample work-up. Gas-phase sequencing of the intact polypeptide chains shows that almost half of the amino acid sequence may be realised by this approach. More comprehensive coverage of the sequence (in excess of 80%) is given by cyanogen bromide digestion of whole blood followed by gas-phase sequencing of the resulting peptide fragments. The simplicity of the cyanogen bromide peptide map, accounting for the entire α- and β-chain sequences, is a distinct advantage of the method. Limitations of characterising aberrant proteins in triple quadrupole instruments are discussed with respect to mass accuracy and the effect of fragment ion charge state on detecting amino acid substitutions.

A method is presented for the simple identification of C-terminal fragment of proteins. The method consists of (i) C-terminal processing of a protein by carboxypeptidase and (ii) comparative peptide mapping of the intact and carboxypeptidase-excised protein after fragmentation by endoproteinase or by chemical cleavage. The peptide mapping was performed by means of high-performance reversed-phase chromatography, where the C-terminal fragment was identified as a peptide peak that was lost or decreased in the carboxypeptidase-excised protein. The C-terminal sequence of the protein could be then determined by sequential Edman degradation of the C-terminal fragment collected from the peptide mapping chromatography. The sensitivity of the method depends solely on the peptide detection and subsequent Edman degradation, currently available techniques of which require a nanomole to subnanomole quantity of protein. The present method can be coupled with conventional carboxypeptidase technology because it utilizes a protein portion remaining after carboxypeptidase digestion while released amino acids are needed in the conventional technique. The method would be particularly valuable in finding a gene probe site for a RNA message coding for the C-terminal portion of a molecule.

Fast atom bombardment (FAB) combined with tandem mass spectrometry has been used to verify the amino acid sequence in Org 2766 [H-Met(O2)-Glu-His-Phe-D-Lys-(Phe-OH)] and its deuterated analogues. It is shown that FAB mass spectrometry in addition to the [M + H]+ ion yields dominantly N-terminal sequence ions and some C-terminal sequence ions. These results together permit determination of the amount of deuterium incorporated and the position of the denterated amino acids in the peptide.

It was shown that the alternate use of p-chloro- and p-bromophenylisothiocyanate for Edman degradation makes it possible to determine the amino acid sequence of peptides more unambigously by mass spectrometry, i.e., by the doublet molecular ions of phenylthiohydantoin derivatives of amino acids with the intensity ratio inherent to the natural abundance of each halogen atom. The amino acid sequence of the shortened peptide could also be surely determined by the deuterium labeled mixed acylation by shifting the “sequence determining peaks” to higher mass region where no interfering peaks for the interpretation of the mass spectra would appear as well as labeling them as doublets with equal intensities by the distance of 2 mass units. Two examples, a dodecapeptide H–(Pro–Pro–Gly)4–OH, and an octapeptide angiotensin II, were presented.

The amino acid sequences of a single peptide and peptide mixture were elucidated from metastable sequence ions produced by collisional activation. All probable sequences were determined by the computer program PAAS. If the constitution of amino acids in the sample peptide is known, a unique peptide sequence was obtained in most cases.

Amino acid thiohydantoins were identified using electron impact and chemical ionization mass spectrometry. Some fragmentations of thiohydantoin ring were also identified. These results suggest that the thiohydantoin method should be useful for the stepwise peptide sequence analysis starting from the carboxyl terminus. The sequence of a model tripeptide was determined by the combination of the thiohydantoin method and mass spectrometry. In the case of peptide which contained a proline residue as their C-terminal peptide, cleavage of the peptide bond cannot be achieved.

The amino acid sequence of a peptide is determined by the combination of the Edman degradation and the field desorption mass spectrometry. The kind of the PTH amino acid is determined by measuring the molecular weights of the residual intact peptides before and after the degradation. The molecular weights are determined from the (M+H)+ peaks of FD mass spectra. The sequence determinations of the peptide fragments in a peptide mixture can be done parallelly and simultaneously by this method.

The combined use of glycinamidation with secondary ion mass spectrometry provides a new strategy for the structural analysis of proteins, especially of acidic ones. Elimination of acid residues by glycinamidation facilitated the tryptic digestion of protein, resulting in an improvement in the quality of peptide mapping on mass spectra. The number of acidic amino acid residues in each peptide was easily calculated from the mass increment by glycinamidation without separation of peptides. Further, the glycinamidation makes easier the structural analysis of variant proteins with an acid/amide substitution by augmenting the molecular weight change.

Reaction-intermediate spectra are reported for peptide mixtures using a hybrid BEQQ and a pentaquadrupole mass spectrometer. In this extended tandem mass spectrometry scan, the protonated molecule and a terminal fragment ion are selected with the first and third mass analyzers, respectively. A subsequent linked scan at constant B/E (hybrid instrument) or a scan of the middle quadrupole (pentaquadrupole instrument) produces the reaction-intermediate spectrum. The resulting spectra are simpler than conventional tandem mass spectrometry daughter-ion spectra and display only sequence ions of one group, viz. A, B and C or X, Y and Z. This information facilitates structural elucidation and is obtainable on nanomole amounts of peptide.

The amino acid sequence of a polypeptide was determined by a new method combined with field desorption (FD) mass spectrometry and Edman degradation. The polypeptide, N-terminal BrCN fragment of Streptomyces erythraeus lysozyme with 55 amino acid residues, was cleaved specifically into several peptide fragments by trypsin and chymotrypsin. The molecular weights of subpeptides were determined by FD mass spectrometry. Peptide mixtures were subjected to Edman degradation successively and PTH amino acids released were measured by high performance liquid chromatography. The amino acid sequence was calculated by a computer program from molecular weights of subpeptides and PTH amino acids.

Products of the gastrin-releasing peptide gene were isolated from culture medium supernatant of a small cell lung cancer line, NCI-H345, by several (high performance liquid chromatography) HPLC steps. The column eluates were monitored by immunoassay and absorbance profiles. Gastrin-releasing peptide was identified in HPLC eluates by a specific radioimmunoassay. Two carboxyl-terminal gastrin-releasing peptide gene-associated peptides were identified by a radioimmunoassay specific for their predicted carboxyl terminus. The amino termini of these two peptides were determined by microsequence analysis. The shorter peptide was revealed to be a fragment of the larger peptide. Expression of an alternate mRNA was shown by isolation and characterization of a novel tetradecapeptide. Amino acid analysis, microsequence analysis, and mass spectral analysis confirmed that the structure was Ser-Leu-Leu-Gln-Val-Leu-Asn-Val-Lys-Glu-Gly-Thr-Pro-Ser. This peptide represents the carboxyl terminus of a peptide resulting from alternate processing of gastrin releasing peptide mRNA. This mRNA contains a 19-base deletion, creating a frame shift. A radioiodinated synthetic analog of this peptide (Tyr-Leu-Val-Asp-Ser-Leu-Leu-Gln-Val-Leu-Asn-Val-Lys-Glu-Gly-Thr-Pro-Ser ) bound specifically to a small cell cancer line with high affinity, suggesting possible biological activity of the isolated peptide.

In order to resolve the uncertainties about the primary structure of human myelin basic protein at residues 45-89, the sequence of this peptide and its tryptic fragments were reinvestigated by fast atom bombardment mass spectrometry. The sequence at positions 77-78 was found to be His-Gly and the sequence at positions 83-84 was shown to be Glu-Asn. The Ser at position 56 was not phosphorylated, whereas the residue at position 46 or 47 showed a heterogeneity of Gly and Ser in this peptide fragment in one of two protein preparations from different patients. These results demonstrate the usefulness of fast atom bombardment mass spectrometry for primary structure information. The corrected sequence of human basic protein peptide 45-89 will permit a more detailed immunochemical analysis of this peptide and its in vivo degradation products.

A graphics display-oriented method for the computer-aided interpretation of the mass spectrum of a peptide in terms of its amino acid sequence is presented. The spectrum is obtained by collision induced decomposition of the protonated molecular ion (M+H)+ of the peptide generated by fast atom bombardment in the first double focussing mass analyzer of a tandem mass spectrometer and the product ion mass spectrum (CID spectrum) recorded by the second double focussing mass analyzer. The algorithm displays all series of peaks differing by the mass of -NH-CHR-CO- for the various amino acids. This display, which is very fast and can be augmented through interrogation by the user, greatly facilitates the determination of the amino acid sequence. The method is demonstrated on the spectra of a undecapeptide of (M+H)+ m/z = 1208.2 and a tetradecapeptide of (M+H)+ m/z = 1758.9.

Peptide A, the C-terminal cyanogen bromide fragment of thioredoxin, was degraded with chymotrypsin and pepsin. Partial sequences of 12 chymotryptic and 6 peptic peptides and the N-terminal tryptic peptide of trifluoro-acetylated peptide A were determined. The results were used to establish the order of the previously described tryptic peptides of peptide A and lead to the complete amino acid sequence of peptide A. Previous experiments had established the amino acid sequence of peptide B, the N-terminal cyanogen bromide fragment of thioredoxin and the present results thus give the complete amino acid sequence of thioredoxin from Escherichia coli B. The molecule contains 108 residues in a single polypeptide chain with a molecular weight of 11,657 as calculated from the sequence. The functional group of the protein occurs in position 32 to 35 and consists of a disulfide bridge formed by two half-cystine residues separated by a glycine and a proline residue. No metals were found as part of the functional group.

Micro-quantities of protein-derived peptides have been converted into N-acetylated permethyl derivatives, and their sequences determined by low-resolution mass spectrometry without prior knowledge of their amino acid compositions or lengths. A new strategy is suggested for the mass spectrometric sequencing of oligopeptides or proteins, involving gel filtration of protein hydrolysates and subsequent sequence analysis of peptide mixtures. Finally, results are given that demonstrate for the first time the use of mass spectrometry for the analysis of a protein-derived peptide mixture, again without prior knowledge of the protein or components within the mixture.

Capillary high-performance liquid chromatography has been coupled on-line with an ion trap storage/reflectron time-of-flight mass spectrometer to perform tandem mass spectrometry for tryptic peptides. Selection and fragmentation of the precursor ions were performed in a three-dimensional ion trap, and the resulting fragment ions were pulsed out of the trap into a reflectron time-of-flight mass spectrometer for mass analysis. The stored waveform inverse Fourier transform waveform was applied to perform ion selection and an improved tickle voltage optimization scheme was used to generate collision-induced dissociation. Tandem mass spectra of various doubly charged tryptic peptides were investigated where a conspicuous y ion series over a certain mass range defined a partial amino acid sequence. The partial sequence was used to determine the identity of the peptide or even the protein by database search using the sequence tag approach. Several peptides from tryptic digests of horse heart myoglobin and bovine cytochrome c were selected for tandem mass spectrometry (MS/MS) where it was demonstrated that the proteins could be identified based on sequence tags derived from MS/MS spectra. This approach was also utilized to identify protein spots from a two-dimensional gel separation of a human esophageal adenocarcinoma cell line.

We describe the design, preparation, and mass-spectrometric characterization of a new recombinant peptide calibration standard with uniform biophysical and ionization characteristics for mass spectrometry. “PAS-cal” is an artificial polypeptide concatamer of peptide cassettes with varying lengths, each composed of the three small, chemically stable amino acids Pro, Ala, and Ser, which are interspersed by Arg residues to allow site-specific cleavage with trypsin. PAS-cal is expressed at high yields in Escherichia coli as a Small Ubiquitin-like MOdifier (SUMO) fusion protein, which is easily purified and allows isolation of the PAS-cal moiety after SUMO protease cleavage. Upon subsequent in situ treatment with trypsin, the PAS-cal polypeptide yields a set of four defined homogeneous peptides in the range from 2 to 8 kDa with equal mass spacing. ESI-MS analysis revealed a conveniently interpretable raw spectrum, which after deconvolution resulted in a very simple pattern of four peaks with similar ionization signals. MALDI-MS analysis of a PAS-cal peptide mixture comprising both the intact polypeptide and its tryptic fragments revealed not only the four standard peptides but also the singly and doubly charged states of the intact concatamer as well as di- and trimeric adduct ion species between the peptides, thus augmenting the observable m/z range. The advantageous properties of PAS-cal are most likely a result of the strongly hydrophilic and conformationally disordered PEG-like properties of the PAS sequences. Therefore, PAS-cal offers an inexpensive and versatile recombinant peptide calibration standard for mass spectrometry in protein/peptide bioanalytics and proteomics research, the composition of which may be further adapted to fit individual needs. Figure ᅟ

A method to correlate the uninterpreted tandem mass spectra of peptides produced under low energy (10–50 eV) collision conditions with amino acid sequences in the Genpept database has been developed. In this method the protein database is searched to identify linear amino acid sequences within a mass tolerance of ± 1 u of the precursor ion molecular weight. A cross-correlation function is then used to provide a measurement of similarity between the mass-to-charge ratios for the fragment ions predicted from amino acid sequences obtained from the database and the fragment ions observed in the tandem mass spectrum. In general, a difference greater than 0.1 between the normalized cross-correlation functions of the first- and second-ranked search results indicates a successful match between sequence and spectrum. Searches of species-specific protein databases with tandem mass spectra acquired from peptides obtained from the enzymatically digested total proteins of E. coli and S. cerevisiae cells allowed matching of the spectra to amino acid sequences within proteins of these organisms. The approach described in this manuscript provides a convenient method to interpret tandem mass spectra with known sequences in a protein database.

The structural relationship between apolipoprotein B-100 (apo-B-100) and apolipoprotein B-48 (apo-B-48) has not been elucidated. A peptide fragment (MDB-18) of approximately 6 kDa was isolated from a tryptic digest of apo-B-100. The sequence of the first 22 amino acids of MDB-18 was determined by Edman degradation. A 15-residue peptide corresponding to this sequence was synthesized by the solid-phase method and was utilized to develop a sequence-specific polyclonal antibody. On immunoblot analysis, the antibody recognized both intact apo-B-100 and apo-B-48. In addition, preincubating the antibody with the synthetic peptide abolished the recognition of both apo-B-100 and apo-B-48. These data are interpreted as indicating that there is an amino acid sequence homology between apo-B-100 and apo-B-48. Since the MDB-18 peptide is located in the carboxyl region of the B-100 molecule, we propose apo-B-100 and apo-B-48 share a common carboxyl region sequence.

In 1950 Edman published a chemical method for the stepwise removal of amino acids from the N-terminus of a peptide.1 This series of reactions has come to be known as the Edman Degradation, and although modifications of this technique have been introduced from time to time, (see below) the Edman Degradation remains, thirty years after its introduction, the only effective chemical means of removing amino acids in a stepwise fashion from a polypeptide chain.

To overcome the limit of the intensity of ions carrying sequence information in structural determinations of peptides by fast-atom-bombardment m.s., we have developed a method that consists in taking spectra of the peptide acid hydrolysates at different hydrolysis times. Peaks correspond to the oligomers arising from the peptide partial hydrolysis. The sequence can then be identified from the structurally overlapping fragments.

A generally applicable strategy has been developed for the primary sequence determination of peptides and proteins containing gamma-carboxyglutamic acid. The intact peptide or protein is either dissolved in, or allowed to come into vapor phase contact with, 0.05 M DCI and then heated in vacuo at 110 degrees C for several hours. Under these conditions gamma-carboxyglutamic acid quantitatively decarboxylates and incorporates two atoms of deuterium per molecule, resulting in the formation of gamma-dideuteroglutamyl residues. Following enzymatic or acidic degradation of the protein the peptide mixture generated is converted (without further isolation of individual peptides) to the N-trifluoroacetylated, O-trimethylsilylated polyamino alcohols and subsequently analyzed by gas chromatography mass spectrometry. Peptide fragments in which gamma-carboxyglutamic acid was present show sequence ions in their mass spectra corresponding to those of glutamic acid, but shifted upwards by 2 amu. This approach has been used to identify the positions of Gla in a tryptic peptide isolated from blood coagulation factor IX, and is currently being employed in the sequence determination of the Gla-containing bone protein osteocalcin.

Collision-activated dissociation of an [M + 2H]2+ ion from a tryptic peptide of molecular weight 2315.3 provides sets of overlapping fragments that allow the amino acid sequence of this peptide to be determined on a triple quadrupole mass spectrometer having a mass range of 1800 Da.