Determination of the sequences of protein-derived peptides and peptide mixtures by mass spectrometry

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.     

Precursor ion scanning and sequencing of arginine-ADP-ribosylated peptide by mass spectrometry

Arginine (Arg)-specific ADP-ribosylation is one of the posttranslational modifications of proteins and is thought to play an important role in reversibly regulating functions of the target proteins in eukaryotes. However, the physiological target protein has not been established. We examined the fragmentation pattern of both ADP-ribosyl-Arg (ADP-R-Arg) and Arg-ADP-ribosylated peptides by quadrupole tandem mass spectrometry and found a specific cleavage of ADP-R-Arg into N-(ADP-ribosyl)-carbodiimide (ADP-R-carbodiimide) and ornithine. Based on this specific fragmentation pattern, we successfully identified the modification site and sequence of Arg-ADP-ribosylated peptide using a two-step collision and showed that ADP-R-carbodiimide is an excellent marker ion for precursor ion scanning of Arg-ADP-ribosylated peptide. We propose that a combination of the precursor ion scanning with ADP-R-carbodiimide as a marker ion and two-step collision is useful in searching for physiological target proteins of Arg-ADP-ribosylation.     

Ion mobility mass spectrometry for peptide analysis

The use of ion mobility mass spectrometry has grown rapidly over the last two decades. This powerful analytical platform now forms an attractive prospect for comprehensive analysis of many different molecular species, including chemically complex biological molecules. This paper describes the application of IM-MS to the study of peptides. We focus on three different ion mobility devices that are most frequently found in tandem with mass spectrometers. These are instruments using linear drift tubes (LDT), those using travelling wave ion guides (TWIGS) and those employing high field asymmetric ion mobility spectrometry (FAIMS). Each technique is described. Examples are given on the use of IM-MS for the determination of peptide structure, the study of peptides that form amyloid fibrils, and the study of complex peptide mixtures in proteomic investigations. We describe and comment on the methodologies used and the outlook for this developing analytical technique.     

Incorporation of tandem mass spectrometric detection to the analysis of peptide mixtures by continuous flow fast atom bombardment mass spectrometry

The ability to acquire structurally informative daughter ion spectra for individual peptides undergoing separation and analysis by continuous flow fast atom bombardment (CF FAB) is demonstrated. To illustrate the potential of this methodology, tryptic and chymotryptic digests of the 29-residue peptide glucagon were analyzed by CF FAB using mass spectrometric and tandem mass spectrometric detection in consecutive analyses. Daughter ion spectra were recorded using B/E linked scans for the major hydrolysis products observed by liquid chromatography/mass spectrometry. The peptide mixtures were separated by gradient capillary high-performance liquid chromatography with the FAB matrix being added post-column using a coaxial flow interface between the column and flow probe. The entire effluent (3 microl min(-1)) was sampled by the mass spectrometer. Results obtained using less than 300 pmol of digested glucagon indicated several advantages to tandem mass spectrometric detection including the ability to confirm identities for products of enzymatic digestion and the potential use of this method for tandem sequence analysis of peptide mixtures.     

Analysis of iodinated peptides by LC-DAD/ESI ion trap mass spectrometry

The analysis of iodinated peptides resulting from chloramine-T (CAT), Iodo-Beads, Iodo-Gen and lactoperoxidase iodination reactions in the preparation of nanomole quantities 125I and 123I labelled tracers is described. Seven different model peptides were evaluated, varying in molecular weight from 294 (LY-dipeptide) to 2518 (obestatin containing 23 amino acid residues). Two different RP-C18 columns were used, each with a different gradient system based on aqueous formic acid and acetonitrile. Electrospray ionization (ESI) ion trap mass spectrometry was used for identification of the chromatographic eluting components of the reaction mixtures, while UV (DAD) served quantitative purposes. Non-, mono-, di-, tri- and tetra-iodinated peptides (respectively NIP, MIP, DIP, 3IP and 4IP) eluted in that order and were well separated from each other. An empirical model was derived. The applicability of this approach was demonstrated by the analysis of different reaction mixtures.     

Assessing data quality of peptide mass spectra obtained by quadrupole ion trap mass spectrometry

An algorithm is introduced to assess spectral quality for peptide CID spectra acquired by a quadrupole ion trap mass spectrometer. The method employs a quadratic discriminant function calibrated with manually classified 'bad' and 'good' quality spectra, producing a single 'spectral quality' score. Many spectra examined that do not have significant matches are assessed to have good spectral quality, indicating that advances in search methods may yield substantial improvements in results.     

Molecular species analysis of lipids by metastable ion mass spectrometry

A convenient universal and fast mass spectrometrical method designed for the molecular species analysis of natural lipids is described. In contrast to the commonly employed procedures the method does not require chemical or enzymatic treatment and does not include chromatographic steps. The method relies on the recognition of ions characteristic of individual molecular species in the mass spectrum of a particular lipid fraction, that is accomplished on the basis of metastable ion spectra. The efficiency of this approach is demonstrated with a variety of natural lipids: triglycerides, glycerophospholipids, sphingomyelin and ornithinolipids. The advantages and limitations of the method as well as possible further developments are discussed.     

Derivatization of hydrophilic peptides for liquid secondary ion mass spectrometry at the picomole level

A simple procedure for preparing alkyl and benzyl esters of peptides is described. The procedure can provide an increase in the secondary ion yield of a factor of 25 or more in the liquid secondary ion mass spectra of hydrophilic peptides. The procedure allows rapid in situ derivatization of, e.g., collected, lyophilized HPLC fractions. No sample transfers are required and excess reagents are easily removed. Mass spectrometry of such fractions is typically required to prepare a mass map of the peptides produced by proteolytic digestion of a protein. However, small hydrophilic peptides are often not detected because their low secondary ion yield. Relative yields of MH+ ions from peptides esterified with various alcohols are compared: methanol, 2-propanol, 1-butanol, 1-hexanol, 1-octanol, and benzyl alcohol. The best combination of ion yield and ease of reagent removal is obtained with 1-hexanol. The degree of improvement depends on the specific peptide; the greatest improvement is generally observed with the most hydrophilic peptides. The procedure does not affect side-chain amides. Partial derivatization is sometimes observed with peptides containing more than one carboxyl group. Hexylation is shown to have a leveling effect on the mass spectra of peptide mixtures, allowing detection of surface-inactive peptides in the presence of surface-active ones. Benzyl alcohol is useful for derivatizing peptides that are not retained or that elute very early from reverse-phase HPLC columns. The derivatives have longer retention times and greater uv molar absorptivity and are more easily detected by subsequent mass spectrometry than the underivatized peptides.     

Developments in mass spectrometry for the analysis of complex protein mixtures.

State-of-the-art proteomics workflows involve multiple interdependent steps: sample preparation, protein-peptide separation, mass spectrometry and data analysis. While improvements in any of these steps can increase the depth and breadth of analysis, advances in mass spectrometry have catalysed many of the most important developments. We discuss common classes of mass analysers and how these analysers are put together to produce some of the most popular mass spectrometry platforms. The capabilities of these platforms determine how they can be used in a variety of common proteomic strategies and, in turn, what types of biological questions can be addressed. Moving forward, powerful new hybrid mass spectrometers and application of emerging types of tandem mass spectrometry promise that our ability to analyse complex mixtures of proteins will continue to advance.     

Lipid fingerprints of intact viruses by MALDI-TOF/mass spectrometry

A number of viruses contain lipid membranes, which are in close contact with capsid proteins and/or nucleic acids and have an important role in the viral infection process. In this study membrane lipids of intact viruses have been analysed by MALDI-TOF/MS with a novel methodology avoiding lipid extraction and separation steps. To validate the novel method, a wide screening of viral lipids has been performed analysing highly purified intact bacterial and archaeal viruses displaying different virion architectures. Lipid profiles reported here contain all lipids previously detected by mass spectrometry analyses of virus lipid extracts. Novel details on the membrane lipid composition of selected viruses have also been obtained. In addition we show that this technique allows the study of lipid distribution easily in subviral particles during virus fractionation. The possibility to reliably analyse minute amounts of intact viruses by mass spectrometry opens new perspectives in analytical and functional lipid studies on a wider range of viruses including pathogenic human ones, which are difficult to purify in large amounts.     

Extraction method for analysis of detergent-solubilized bacteriorhodopsin and hydrophobic peptides by electrospray ionization mass spectrometry

The analysis of integral membrane proteins or transmembrane peptides by electrospray ionization mass spectrometry (ESI-MS) is difficult since detergents, used to solubilize these hydrophobic proteins and peptides, severely suppress analyte ion formation. This problem has been addressed previously by precipitating the protein, removing the detergent, and resolubilizing the protein in a nonpolar solvent. Here, we demonstrate a method that avoids protein precipitation and resolubilization. Detergent-solubilized bacteriorhodopsin is extracted into a nonpolar solvent phase by adding a chloroform/methanol/water solvent mixture to the aqueous detergent solution. ESI mass spectra of the nonpolar, chloroform-rich phase were dominated by peaks due to bacterioopsin. Bacterioopsin precursors with partially cleaved leader sequences were seen in all mass spectra. Additional peaks were likely due to intact bacteriorhodopsin, i.e., bacterioopsin with the retinal prosthetic group attached, and to bacterioopsin associated with lipid molecules. A separation process that occurred in the fused-silica capillary leading to the electrospray tip was essential for obtaining ESI mass spectra of bacterioopsin. The extraction-into-chloroform procedure also worked well with hydrophobic, transmembrane-type peptides that were insoluble in other electrospray solvents, including 100% formic acid, and the method has application to transmembrane peptides formed from digests of integral membrane proteins.     

Quantitative analysis of tryptic protein mixtures using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

For the first time, quantitative analysis of tryptic protein mixtures, labeled with Quantification-Using-Enhanced-Signal-Tags (QUEST)-markers, were performed with electrospray ionization and a 9.4 T Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer. Coupling a High-Pressure Liquid Chromatography (HPLC) separation step prior to mass analysis resulted in an increased amount of identified labeled tryptic peptides. The range for the determined intensity ratios of two peptides in a labeled pair was large, but the obtained median intensity ratio correlated very well with the corresponding concentration ratio. This method can be used for observing protein dynamics in a specific cell type, tissue, or in body fluids.     

The analysis of multiple O-phosphoseryl-containing peptides by fast atom bombardment mass spectrometry

Summary Positive and negative ion FAB mass spectrometry were found to be useful for the structural analysis of phosphorylated peptides containing multiple O-phosphoseryl residues. The positive ion FAB mass spectra obtained for Ac-Ser(P)-Ser(P)-NHMe and Ac-Ser(P)-Ser(P)-Ser(P)-NHMe showed that -eliminative loss of H₃PO₄ from the Ser(P)-residue was a major event in the fragmentation of the two phosphopeptides and that successive losses of H₃PO₄ from the [M+H]⁺ ion occurred when the Ser(P)-cluster was located at the N-terminus. In contrast, the FAB mass spectrum of Ac-Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe showed only a single loss of H₃PO₄ from the [M+H]⁺ ion, with further losses of H₃PO₄ from internal Ser(P)-residues only occurring when fragmentation of the parent phosphopeptide generated daughter fragments that contained (part of) an N-terminal Ser(P)-residue. Negative ion FAB mass spectrometry also proved useful for the structural analysis of the three Ser(P)-peptides and showed high-intensity [M-H]^- ions along with minor [M-H-80]^- fragment ions.Abbreviations Ac acetyl - Ala dehydroalanyl - FAB-MS fast atom bombardment mass spectrometry - LSIMS liquid secondary ion mass spectrometry - NHMe N-methylamide - Ser(P) O-phosphoseryl - Thr(P) O-phosphothreonyl     

Nanostructure-initiator mass spectrometry: a protocol for preparing and applying NIMS surfaces for high-sensitivity mass analysis

Nanostructure-initiator mass spectrometry (NIMS) is a new surface-based MS technique that uses a nanostructured surface to trap liquid ('initiator') compounds. Analyte materials adsorbed onto this 'clathrate' surface are subsequently released by laser irradiation for mass analysis. In this protocol, we describe the preparation of NIMS surfaces capable of producing low background and high-sensitivity mass spectrometric measurement using the initiator compound BisF17. Examples of analytes that adsorb to this surface are small molecules, drugs, lipids, carbohydrates and peptides. Typically, NIMS is used to analyze samples ranging from simple analytical standards and proteolytic digests to more complex samples such as tissues, cells and biofluids. Critical experimental considerations of NIMS are described. Specifically, NIMS sensitivity is examined as a function of pre-etch cleaning treatment, etching current density, etching time, initiator composition, sample concentration, sample deposition method and laser fluence. Typically, NIMS surface preparation can be completed in less than 2 h. Subsequent sample preparation requires 1-5 min, depending on sample deposition method. Mass spectrometric data acquisition typically takes 1-30 s per sample.     

Analysis of phosphorylated proteins and peptides by mass spectrometry

Phosphorylation on serine, threonine and tyrosine residues is an extremely important modulator of protein function. Therefore, there is a great need for methods capable of accurately elucidating sites of phosphorylation. Although full characterization of phosphoproteins remains a formidable analytical challenge, mass spectrometry has emerged as an increasingly viable tool for this task. This review summarizes the methodologies currently available for the analysis of phosphoproteins by mass spectrometry, including enrichment of compounds of interest using immobilized metal affinity chromatography and chemical tagging techniques, detection of phosphopeptides using mass mapping and precursor ion scans, localization of phosphorylation sites by peptide sequencing, and quantitation of phosphorylation by the introduction of mass tags. Despite the variety of powerful analytical methods that are now available, complete characterization of the phosphorylation state of a protein isolated in small quantities from a biological sample remains far from routine.