Detection and characterization of native β‐endorphin1–31 in bovine pituitary using electrospray ionization, liquid secondary ion and tandem mass spectrometry
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Abstract The molecular ion of intact β‐endorphin bovine, 1–31 (BE), which was extracted from bovine pituitary, was determined by electrospray ionization mass spectrometry. Liquid secondary ion mass spectrometry determined the molecular masses of three peptides produced by trypsin digestion of BE, and tandem mass spectrometry was used to determine the amino acid sequence of the tryptic peptide BE 20–24 . These data, in combination, were used to characterize BE in bovine pituitary.Keywords:
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INTRODUCTION. PREFACE. SECTION I: IONIZATION METHODS. 252Cf PLASMA DESORPTION METHODS. Fundamental Aspects of Protein Mass Spectrometry Using 252-Californium Plasma Desorption. Plasma Desorption Mass Spectrometry of Peptides and Peptide Conjugates. The Analysis of Synthetic Peptides and Proteins. Analysis of Peptides and Proteins by Plasma Desorption Mass Spectrometry. LASER-INDUCED MULTIPHOTON IONIZATION. Laser-Induced Multiphoton Ionization of Peptides in Supersonic Beam/Mass Spectrometry. SECTION II: INSTRUMENTAL DEVELOPMENTS. MAGNETIC SECTOR INSTRUMENTS. The Molecular Weight Determination of Large Peptides by Magnetic Sector Mass Spectrometry. Four-Sector Tandem Mass Spectrometry of Peptides. QUADRUPOLE FOURIER TRANSFORM. Peptide Sequence Analysis by Triple Quadrupole and Quadrupole Fourier Transform Mass Spectrometry. TIME-OF-FLIGHT INSTRUMENTS. Correlation Measurements in a Reflecting Time-of-Flight Mass Spectrometer. SECTION III: ANALYSIS OF PEPTIDES. SAMPLE PREPARATION. Sample Preparation and Matrix Selection for Analysis of Peptides by FAB and Liquid SIMS. LC-MS ANALYSIS. On-Line Methods for Peptide Analysis by Continuous-Flow FABMS. ANALYSIS OF PROTEIN PRODUCTS. Investigation of Amino Acid Mutation by High Resolution Mass Spectrometry. The Mass Spectral Analysis of Hemoglobin Variants. PROTEIN CROSS-LINKAGES. Detection and Location of Disulfide Bonds in Proteins by Mass Spectrometry. PEPTIDE INTERACTIONS WITH METAL IONS. Tandem Mass Spectrometry for Determining the Amino Acid Sequence of Cyclic Peptides and for Assessing Interactions of Peptides and Metal Ions. PERMETHYLATED PEPTIDES. Analysis of Permethylated Peptides by Mass Spectrometry. NEUROPEPTIDES. Applications of Mass Spectrometry for Characterization of Neuropeptides. Peptide-Charting Applied to Studies of Precursor Processing in Endocrine Tissues. QUANTIFICATION OF NEUROPEPTIDES. Mass Spectrometry of Biologically Important Neuropeptides
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Fast atom bombardment
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Mass Spectrometry (MS) allows the analysis of proteins and peptides through a variety of methods, such as Electrospray Ionization-Mass Spectrometry (ESI-MS) or Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). These methods allow identification of the mass of a protein or a peptide as intact molecules or the identification of a protein through peptide-mass fingerprinting generated upon enzymatic digestion. Tandem mass spectrometry (MS/MS) allows the fragmentation of proteins and peptides to determine the amino acid sequence of proteins (top-down and middle-down proteomics) and peptides (bottom-up proteomics). Furthermore, tandem mass spectrometry also allows the identification of post-translational modifications (PTMs) of proteins and peptides. Here, we discuss the application of MS/MS in biomedical research, indicating specific examples for the identification of proteins or peptides and their PTMs as relevant biomarkers for diagnostic and therapy.
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Protein mass spectrometry refers to the application of mass spectrometry to study the proteins and nowadays it is introduced in proteomies science as well as pharmaceutical biotechnology. Mass spectrometry is an important emerging method for ionization of whole proteins are electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI). In keeping with the performance and mass range of available mass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins are ionized by either of the two techniques described above, and then introduced to a mass analyzer. This approach is referred to as “top-down” strategy of protein analysis. In the second, proteins are enzymatically digested into smaller peptides using a protease such as trypsin. Subsequently these peptides are introduced into the mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry. Hence this latter approach (also called “bottom up” proteomies) uses identification at the peptide level to infere the existence of proteins.
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Mass spectrometry has become an important technique to correlate proteins to their genes. This has been achieved, in part, by improvements in ionization and mass analysis techniques concurrently with large-scale DNA sequencing of whole genomes. Genome sequence information has provided a convenient and powerful resource for protein identification using data produced by matrix-assisted laser desorption/ionization time-of-flight (MALDI/TOF) and tandem mass spectrometers. Both of these approaches have been applied to the identification of electrophoretically separated protein mixtures. New methods for the direct identification of proteins in mixtures using a combination of enzymatic proteolysis, liquid chromatographic separation, tandem mass spectrometry and computer algorithms which match peptide tandem mass spectra to sequences in the database are also emerging. This tutorial review describes the principles of ionization and mass analysis for peptide and protein analysis and then focuses on current methods employing MALDI and electrospray ionization for protein identification and sequencing. Database searching approaches to identify proteins using data produced by MALDI/TOF and tandem mass spectrometry are also discussed. © 1998 John Wiley & Sons, Ltd.
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A method for the characterization of modifications of low molecular weight proteins (<20 kDa) extracted from a microorganism based on the use of multiple separation tools and mass spectrometric techniques is described. In this method, intact proteins from cell extracts are first separated and fractionated by liquid chromatography (LC). Individual fractions are then analyzed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) to provide intact protein mass information. The fractions are further characterized by using trypsin digestion and LC electrospray ionization (ESI) MS/MS analysis of the resultant peptides to identify the proteins. Gel electrophoresis of a fraction is also carried out to estimate the molecular masses of the proteins. The gel bands are identified by in-gel digestion and peptide mass mapping and sequencing using MALDI-MS and MALDI-MS/MS. The combined information generated from these experiments is interpreted for detecting and characterizing modified proteins. This method has been developed and applied to the analysis of posttranslational modifications (PTMs) of low-mass proteins (5–20 kDa) extracted from a relatively well-characterized microorganism, Escherichia coli. Using this method, not only previously reported PTMs involving acetylation, methylation, oxidation, and the removal of signal peptides, but also two novel PTMs, namely loss of N-terminal Met-Thr-Met (MTM) and hydroxylation of arginine, were identified. It is envisaged that this method should be applicable to other relatively simple microorganisms for the discovery of new PTMs.Key words: top-down proteomics, protein modification, HPLC, gel electrophoresis, tandem mass spectrometry.
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Mass spectrometry has become an important technique to correlate proteins to their genes. This has been achieved, in part, by improvements in ionization and mass analysis techniques concurrently with large-scale DNA sequencing of whole genomes. Genome sequence information has provided a convenient and powerful resource for protein identification using data produced by matrix-assisted laser desorption/ionization time-of-flight (MALDI/TOF) and tandem mass spectrometers. Both of these approaches have been applied to the identification of electrophoretically separated protein mixtures. New methods for the direct identification of proteins in mixtures using a combination of enzymatic proteolysis, liquid chromatographic separation, tandem mass spectrometry and computer algorithms which match peptide tandem mass spectra to sequences in the database are also emerging. This tutorial review describes the principles of ionization and mass analysis for peptide and protein analysis and then focuses on current methods employing MALDI and electrospray ionization for protein identification and sequencing. Database searching approaches to identify proteins using data produced by MALDI/TOF and tandem mass spectrometry are also discussed. © 1998 John Wiley & Sons, Ltd.
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Abstract Mass spectrometry is playing an increasingly important role in the characterization and quantification of peptides and proteins. In fact, more conventional approaches to characterization, including techniques such as Edman sequencing and amino acid ( AA ) analysis, have been largely displaced. Mass analysis – typically following either matrix‐assisted laser desorption ionization ( MALDI ) or electrospray ionization ( ESI ) – offers rapid molecular mass determinations with high mass accuracy on subpicomole amounts of material and information can be obtained on the constituents of complex mixtures. The application of tandem mass spectrometry ( MS/MS ) allows fragmentation of peptide ions, and also of proteins. The MS/MS spectra of peptides and proteins can be interpreted de novo or matched against database entries to provide partial amino acid sequence information and peptide/protein identification. Both peptide‐centric (bottom‐up) and protein‐centric (top‐down) approaches to protein characterization have been adopted and are now routinely applied. Mass spectrometry has also been pivotal to the development, evolution, and wide application of proteomics: i.e. the large‐scale identification of the proteins present in a complex biological sample. Proteomics is now routinely applied to characterize and quantify the constituents of complex biological tissues and fluids. This article describes the available approaches to peptide and protein characterization based on mass spectrometry ( MS ), the constantly evolving analytical methods for proteomics, and available strategies for protein quantification.
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Mass spectrometry (MS) has emerged as a sensitive, versatile, and rapid method for protein identification, following the advent of electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The advantages of MALDI-MS over ESI-MS include its relatively high tolerance to contamination from biological matrices, its high sensitivity, the relative ease of interpreting spectra from mixtures, and the formation of singly protonated molecular ions for tandem analysis. Peptide fingerprint mass mapping and partial peptide sequencing using post-source decay and ladder sequencing by MALDI-MS in combination with algorithms for sequence database interrogation have the potential for identification and structural investigation of proteins. This unit describes in-gel digestion for peptide mass mapping of picomole to subpicomole quantities of protein derived from Coomassie- or silver-stained polyacrylamide gels. After digestion, the peptides are extracted from the gel and mass analyzed.
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Abstract Mass spectrometry is playing an increasingly important role in the characterization and quantification of peptides and proteins. In fact, more conventional approaches to characterization, including techniques such as Edman sequencing and amino acid (AA) analysis, have been largely displaced. Mass analysis — following either matrix‐assisted laser desorption ionization (MALDI) or electrospray ionization (ESI) — offers rapid molecular mass determinations with high mass accuracy on subpicomole amounts of material and information can be obtained on the constituents of complex mixtures. The application of tandem mass spectrometry (MS/MS) allows fragmentation of peptide ions, and of proteins with some enhancements too. The MS/MS spectra of peptides and proteins can interpreted de novo or matched against database entries to provide partial amino acid sequence information and peptide/protein identification. Both peptide‐centric (bottom‐up) and protein‐centric (top‐down) approaches to protein characterization have been adopted and are now routinely applied. Mass spectrometry has also been pivotal to the development and wide application of proteomics: i.e. the large‐scale identification of the proteins present in a complex biological sample. Proteomics is now routinely applied to characterize and quantify the constituents of complex biological tissues and fluids. This entry describes the available approaches to peptide and protein characterization based on mass spectrometry, the constantly evolving analytical methods for proteomics, and available strategies for protein quantification.
Bottom-up proteomics
Tandem mass tag
Top-down proteomics
Isobaric labeling
Peptide mass fingerprinting
Protein sequencing
Edman degradation
Proteome
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