Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

High-sensitivity, high-throughput analysis of proteins for proteomics studies is usually performed by polyacrylamide gel electrophoresis in combination with mass spectrometry. However, the quality of the data obtained depends on the in-gel digestion procedure employed. This work describes an improvement in the in-gel digestion efficiency for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis. A dramatic improvement in the coverage of tryptic peptides was observed when n-octyl glucoside was added to the buffer. Whole cell extracted proteins from S. cerevisiae were separated by two-dimensional gel electrophoresis and stained with silver. Protein spots were identified using our improved in-gel digestion method and MALDI-TOFMS. In addition, the mass spectra obtained by using the matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) were compared with those obtained using 2,5-dihydroxybenzoic acid (DHB). The DHB matrix usually gave more peaks, which led to higher sequence coverage and, consequently, to higher confidence in protein identification. This improved in-gel digestion protocol is simple and useful for protein identification by MALDI-TOFMS.     

Analysis of intact proteins from cerebrospinal fluid by matrix-assisted laser desorption/ionization mass spectrometry after two-dimensional liquid-phase electrophoresis

A novel combination of methods, two-dimensional liquid-phase electrophoresis (2D-LPE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), have been used for the analysis of intact brain-specific proteins in cerebrospinal fluid (CSF). 2D-LPE is especially useful for isolating proteins present in low concentrations in complex biological samples. The proteins are separated in the first dimension by liquid-phase isoelectric focusing (IEF) in the Rotofor cell and in the second dimension by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in the Preparative cell. The removal of SDS by chloroform/methanol/water, followed by sample preparation with the addition of n-octylglucoside, easily interfaced 2D-LPE with MALDI-TOFMS for analysis of intact proteins. Further characterization by proteolytic digestion is also demonstrated. The knowledge of both the molecular weights of the protein and of the proteolytic fragments obtained by peptide mapping increases specificity for protein identification by searching in protein sequence databases. Two brain-specific proteins in human CSF, cystatin C and transthyretin, were isolated in sufficient quantity for determination of the mass of the whole proteins and their tryptic digest by MALDI-TOFMS. This approach simplified the interface between electrophoresis and MALDI-TOFMS.     

Identification of low molecular weight proteins isolated by 2-D liquid separations

Proteins with molecular mass (M(r)) <20 kDa are often poorly separated in 2-D sodium dodecyl sulfate polyacrylamide gel electrophoresis. In addition, low-M(r) proteins may not be readily identified using peptide mass fingerprinting (PMF) owing to the small number of peptides generated in tryptic digestion. In this work, we used a 2-D liquid separation method based on chromatofocusing and non-porous silica reversed-phase high-performance liquid chromatography to purify proteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis and protein identification. Several proteins were identified using the PMF method where the result was supported using an accurate M(r) value obtained from electrospray ionization TOFMS. However, many proteins were not identified owing to an insufficient number of peptides observed in the MALDI-TOF experiments. The small number of peptides detected in MALDI-TOFMS can result from internal fragmentation, the few arginines in its sequence and incomplete tryptic digestion. MALDI-QTOFMS/MS can be used to identify many of these proteins. The accurate experimental M(r) and pI confirm identification and aid in identifying post-translational modifications such as truncations and acetylations. In some cases, high-quality MS/MS data obtained from the MALDI-QTOF spectrometer overcome preferential cleavages and result in protein identification.     

Identification of proteins by combination of size-exclusion chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and comparison of some desalting procedures for both intact proteins and their tryptic digests

Separation of a protein mixture by size-exclusion chromatography (SEC) was combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Identification of proteins in the collected fractions was performed both as intact proteins by MALDI-TOFMS and using peptide mass fingerprinting (PMF) after their digestion with trypsin. The presence of salts mostly disturbs the MALDI-TOFMS signal and, therefore, proper purification or desalting procedures must be employed. Four desalting procedures (desalting column packed with Sephadex G-100, on-target washing, centrifugal filter devices and ZipTip C(18)) for purification of fractions of proteins separated by SEC and their tryptic digests prior to determination of their exact molecular masses by MALDI-TOFMS were compared. In the case of intact proteins, the experiments showed that the best desalting procedures are the use of ZipTip C(18) pipette tips and Ultrafree CL centrifugal filter devices. The peptide digests can be purified by using ZipTip C(18) pipette tips or on-target washing when both of these procedures provide similar results. On-target washing can be used as a simple procedure to improve the mass spectra of salt-containing samples. Analyses of the droplets collected after the on-target washing show losses of sample and matrix caused by dissolution of these compounds during this procedure. Further, it was found that protein identification based on PMF is more sensitive than analyses of intact proteins and that multiple on-target washing is very advantageous for analyses of peptide mixtures with a high content of salts.     

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.     

Characterisation of standard tempera painting layers containing proteinaceous binders by pyrolysis (/methylation)-gas chromatography-mass spectrometry

Summary Thirty standard painting layers were analysed by pyrolysis-gas chromatography/mass spectrometry (Py-GC-MS) and by Py-GC-MS in the presence of tetramethylammonium hydroxide (pyrolysis/methylation). Painting layers were prepared according to Renaissance recipes for tempera, employing proteinaceous binders (egg, glue and casein) and six different pigments. Thermal degradation products of proteins, carbohydrates and lipids were selected for semiquantitative analysis based on single/summed ion monitoring (SIM) mode. The relative distribution of these products was used to characterise binding media for the purpose of their identification in painting layers.     

Exopeptidase degradation for the analysis of phosphorylation site in a mono-phosphorylated peptide with matrix-assisted laser desorption/ionization mass spectrometry.

The utility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) coupled with a peptide ladder sequencing method employing exopeptidase degradation for the analysis of phosphorylation site in a mono-phosphorylated peptide is investigated. MALDI-TOFMS analysis of time-dependent exopeptidase digestion using carboxypeptidase W and aminopeptidase M of the mono-phosphorylated 33-48 fragment isolated from a beta-casein tryptic digestion mixture allowed for the sequencing analysis from both the C-terminus and N-terminus. Negative ion detection MALDI-TOFMS made it possible to clearly measure the peptide ladder of mono-phosphorylated peptide by the strong negative charge localized at the phosphoric acid group. Since exopeptidase activity was suppressed by the existence of a phosphorylated amino acid residue, the termination exopeptidase degradation therefore suggested the existence of a phosphorylated amino acid residue at that site. This peptide ladder sequencing method using exopeptidases was effective for the identification of the site of a phosphorylated amino acid residue by a simple MALDI-TOFMS analysis in the negative ion detection mode.     

Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol-cytochrome c reductase

Complex III of the mitochondrial electron transport chain, ubiquinol-cytochrome c reductase, was isolated by blue native polyacrylamide gel electrophoresis. Ten of the 11 polypeptides present in this complex were detected directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following electroelution of the active complex. Tryptic and chymotryptic digestion of the complex permit the identification of specific peptides from all of the protein subunits with 70% coverage of the 250 kDa complex. The mass of all 11 proteins was confirmed by second dimension Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and elution of the separated polypeptides. Additionally, the identity of the core I, core II, cytochrome c and the Rieske iron-sulfur protein were confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) characterization of the peptides generated by in-gel trypsin digestion of the SDS-PAGE separated proteins. The methodology demonstrated for analyzing this membrane-bound electron transport complex should be applicable to other membrane complexes, particularly the other mitochondrial electron transport complexes. The MS analysis of the peptides obtained by in-gel digestion of the intact complex permits the simultaneous characterization of the native proteins and modifications that contribute to mitochondrial deficits that have been implicated as contributing to pathological conditions.     

Capillary electrophoresis off-line matrix-assisted laser desorption/ionisation mass spectrometry of intact and digested proteins using cationic-coated capillaries

Capillary electrophoresis (CE) was coupled off-line with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the analysis of proteins and peptides. CE fractions were collected directly on a matrix-coated MALDI target, using a sheath-flow interface. Protein adsorption during CE separations was prevented by coating the capillaries with the physically adsorbed, cationic polymer PolyE-323. The CE/MALDI-MS system was used for the analysis of model proteins and peptides at physiological pH as well as analysis of proteins in tear fluid. Moreover, tryptic on-target digestion of the collected protein fractions, with subsequent MALDI-MS and MS/MS peptide analysis, was demonstrated.     

On-column digestion of proteins in aqueous-organic solvents

Proteolytic digestion is an important step in protein identification by peptide mass mapping and tandem mass spectrometry (MS/MS)-based peptide sequencing. Traditional methods of protein digestion require extended incubation times and have difficulty with proteolytically resistant proteins. Here, we describe a method in which a protein solution was combined with a mixed aqueous-organic solution (methanol, isopropanol, or acetonitrile) and passed through a microcolumn containing immobilized trypsin. Myoglobin sequence coverage was high (>85%) in all three solvents, and differences in spectra were seen among the different solution conditions. Notably, methanol-based digestions produced fewer missed cleavages while acetonitrile-based digestions produced the most peptides and the most intense mass spectra. Flow rates through the column were varied from 0.5 to 15 micro L/min, corresponding to column residence times of 78 and 2.6 s, respectively. All flow rates produced high sequence coverage of myoglobin, although, at higher flow rates, more missed cleavages were observed. No significant increase in undigested myoglobin was observed with flow rates up to 15 micro L/min. The described method was applied to the digestion of human transferrin (hTf), a proteolytically resistant protein. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis detected 42 peptides covering 46% of the hTf sequence. The traditional aqueous method resulted in 12 peptides (8% sequence coverage) only when high concentrations of trypsin were used. Lastly, digestion of low nanomolar myoglobin was shown to produce detectable peptides and resulted in a correct database hit. Thus, we demonstrate a method that is capable of rapid on-line digestion, thereby lending itself to high-throughput identification of proteins.     

Diagnostic protein discovery using proteolytic peptide targeting and identification

Plasma protein profiling with mass spectrometry is currently being evaluated as a diagnostic tool for cancer and other diseases. These experiments consist of three steps: plasma protein fractionation, analysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), and comparisons of the MALDI profiles to develop diagnostic fingerprints using bioinformatic techniques. While preliminary results appear promising in small sample groups, the method is limited by the sensitivity of MALDI-MS for intact proteins, the limited mass range of MALDI-MS, and difficulties associated with isolating individual proteins for identification to validate the diagnostic fingerprint. Here we present an alternative and improved method directed toward diagnostic protein discovery, which incorporates proteolytic peptide profiling, bioinformatic targeting of ion signals, and MALDI tandem mass spectrometry (MS/MS) peptide sequencing, rather than fingerprinting. Pancreatic cancer patients, pancreatitis patients, and controls are used as the model system. Profiling peptides after enzymatic digestion improves sensitivity and extends the accessible protein molecular weight range when compared to intact protein profiling. The first step is to extract and fractionate the proteins from plasma. Each fraction is digested with trypsin and subsequently analyzed by MALDI-MS. Rather than using bioinformatic analysis as a pattern-matching technique, peptides are targeted based on the disease to control peak intensity ratios measured in the averages of all mass spectra in each group and t-tests of the intensity of each individual peak. The targeted peptide ion signals are subsequently identified using MALDI-MS/MS in quadrupole-TOF and tandem-TOF instruments. This study found not only the proteins targeted and identified by a previous protein profiling experiment, but also detected additional proteins. These initial results are consistent with the known biology of pancreatic cancer or pancreatitis, but are not specific to those diseases.     

Protein profiles and identification of high performance liquid chromatography isolated proteins of cancer cell lines using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been used to rapidly profile the protein content of human cell lysates from MCF-10 cell and variant lines. The method was used to study the protein profiles of these cells as they progressed from normal breast epithelium to fully malignant cells. Distinct differences in the protein profiles were observed with progression, and specific proteins associated with carcinogenesis (p53, c-myc, and c-erbB-2) were heavily expressed in these cells as detected by MALDI-TOFMS. These proteins were also isolated using non-porous reversed-phase high performance liquid chromatography (NP-RP-HPLC) and mass analyzed by MALDI-TOFMS to provide molecular weight information without interference from other proteins in the whole cell lysates, and to avoid suppression effects in mixtures of proteins detected by MALDI-TOFMS. In order to confirm the identity of these oncoproteins, the cell lysates were subjected to one-dimensional (1-D) gel separation and subsequently electroblotted onto a poly(vinylidene difluoride) (PVDF) membrane for further analysis. Trypsin and cyanogen bromide digestions were performed on these proteins eluted from excised PVDF bands which were then analyzed by MALDI-TOFMS. The identity of these proteins was confirmed by database matching procedures.     

Identification of proteinaceous binders used in artworks by MALDI-TOF mass spectrometry

Proper identification of proteinaceous binders in artworks is essential for specification of the painting technique and thus also for selection of the restoration method; moreover, it might be helpful for the authentication of the artwork. This paper is concerned with the optimisation of analysis of the proteinaceous binders contained in the colour layers of artworks. Within this study, we worked out a method for the preparation and analysis of solid samples from artworks using tryptic cleavage and subsequent analysis of the acquired peptide mixture by matrix-assisted laser desorption/ionisation time of flight mass spectrometry. To make this approach rational and efficient, we created a database of commonly used binders (egg yolk, egg white, casein, milk, curd, whey, gelatine, and various types of animal glues); certain peaks in the mass spectra of these binders, formed by rich protein mixtures, were matched to amino acid sequences of the individual proteins that were found in the Internet database ExPASy; their cleavage was simulated by the program Mass-2.0-alpha4. The method developed was tested on model samples of ground layers prepared by an independent laboratory and then successfully applied to a real sample originating from a painting by Edvard Munch.     

Primary structural determination of N-terminally blocked peptides from the bark of Eucommia ulmoides Oliv by mass spectrometric analysis

Sequencing of N-terminally blocked proteins/peptides is a challenge since these molecules inhibit processing by Edman degradation. By using high accuracy Fourier transform ion cyclotron resonance (FTICR) tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), the primary structures of two novel N-terminally blocked antifungal peptides (EAFP1 and EAFP2), purified from the bark of Eucommia ulmoides Oliv, have been determined. The results show that the high mass accuracy provided by FTICR mass spectrometry is effective to determine the N-terminally blocking group, and can simplify the task of spectral interpretation and improve the precision of sequence determination. The combination of MALDI-TOFMS with carboxyl peptidase Y digestion was used to determine the C-terminal 36- and 27-residue sequences of EAFP1 and EAFP2, respectively, to provide the sequence linkage information for tryptic fragments. Compared with traditional peptide chemistry the advantage of mass spectrometric techniques is their simplicity, speed and sensitivity.     

Silver stain removal using H2O2 for enhanced peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Hydrogen peroxide solutions are reported for the removal of silver stain from proteins isolated in polyacrylamide gels. Removal of silver stain prior to in-gel digestion is shown to enhance sensitivity and sequence coverage of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) peptide mass maps. The rate of silver removal using H2O2 is influenced by H2O2 concentration and increases with increasing pH. The presence of complexation reagents such as ammonia from mass spectrometry compatible ammonium bicarbonate solutions enhances the efficiency and speed of H2O2-mediated silver removal. H2O2-mediated silver removal using the described procedure does not appear to have any detrimental effects on proteins but is observed to produce a slightly elevated level of methionine oxidization over that usually observed in in-gel tryptic digestion.     

Charge derivatization by 4-sulfophenyl isothiocyanate enhances peptide sequencing by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

High-sensitivity, rapid identification of proteins in proteomic studies normally uses a combination of one- or two-dimensional electrophoresis together with mass spectrometry. The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) have increased its application in recent years. The most common method of 'peptide fingerprinting' often may not provide robust identification. Normally additional sequence information by post-source decay (PSD) MALDI-TOFMS provides additional constraints for database searches to achieve highly confident results. Here we describe a derivatization procedure to facilitate the acquisition of such sequence information. Peptide digests from a skin-expressed protein were modified with 4-sulfophenyl isothiocyanate. The resulting peptides carry a fixed negative charge at the N-terminal end and the resulting PSD spectrum is dominated by C-terminal y-type ions. The sequence information in most cases can be obtained manually or with simple programming tools. Methods of optimizing the procedure and increasing the sensitivity are discussed.     

Conductive carbon tape as a sample platform for microwave-based MALDI MS detection of proteins and phosphoproteins

In this study, we developed a novel microwave-assisted protein preparation and digestion method for matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry analysis and identification of proteins that involves using conductive carbon tape as a sample platform for sample preparation (reduction and alkylation) and digestion under microwave heating and as a plate for MALDI analysis. This method allows for the enzymatic digestion products of proteins to be directly analyzed by MALDI mass spectrometry and results in a marked reduction in sample loss. Our protocol requires only a small volume (1 μL) of reaction solvent, which increases the frequency of enzyme-to-protein contact, thereby resulting in more efficient digestion of sample than conventional in-solution digestion methods. To test this protocol, we used magnetic iron (II, III) oxide nanoparticles as concentrating probes to enrich phosphopeptides from a mixture of peptides in enzymatically digested protein samples. We found that the one-pot on-tape-based protein preparation and digestion under microwave heating combined with the on-tape-based enrichment method not only dramatically reduced the time required for phosphopeptides analysis but also allowed for the simultaneous identification of phosphoproteins. The advantages of our protocol include ease of use, high digestion efficiency, high specificity, and rapid (15 min) identification of proteins and enrichment of phosphopeptides in a mixture of enzymatically digested protein samples.     

The use of nonpolar matrices for matrix-assisted laser desorption/ionization mass spectrometric analysis of high boiling crude oil fractions

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) with nonpolar matrices has been investigated for its applicability to the characterization of atmospheric resid crude oil fractions. The data obtained by use of nonpolar matrices was compared with that from polar matrices as well as from direct LDI-MS and field ionization mass spectrometry. Nonpolar matrices, such as anthracene or 9-cyanoanthracene, yield only a single radical molecular cation upon LDI. Thus, no interfering matrix-related ions are present during the MALDI-TOFMS analysis of the crude oil sample. Nonpolar matrices yield molecular mass distributions from linear mode MALDI-TOFMS that are comparable to distributions found with LDI-MS. An advantage of nonpolar matrices is the increased production of analyte ions, which allows reflectron mode MALDI-TOFMS to be performed. Nonpolar matrices are also shown to be less sensitive to solvent and sample preparation conditions than conventional polar matrices. These results suggest that nonpolar matrices may be favorable alternatives to more traditional polar or acidic matrices commonly used in the MALDI mass spectral characterization of crude oil related samples.     

A simple protocol for Matrix Assisted Laser Desorption Ionization- time of flight-mass spectrometry (MALDI-TOF-MS) analysis of lipids and proteins in single microsamples of paintings

A simple protocol, based on Bligh–Dyer (BD) extraction followed by MALDI-TOF-MS analysis, for fast identification of paint binders in single microsamples is proposed. For the first time it is demonstrated that the BD method is effective for the simultaneous extraction of lipids and proteins from complex, and atypical matrices, such as pigmented paint layers. The protocol makes use of an alternative denaturing anionic detergent (RapiGest™) in order to improve efficiency of protein digestion and purification step. Detection of various lipid classes, such as triacylglycerols (TAGs) and phospholipids (PLs), and their oxidation by-products was accomplished, whereas proteins could be identified by peptide mass fingerprinting. The effect of pigments on ageing of lipids and proteins was also investigated.     

Behaviour of phospholipids in analytical reactive pyrolysis

Checking for the presence of egg in a painting layer allows to decide whether or not it is a tempera. Several already assessed analytical techniques may be used to perform the chemical analysis for the detection of egg in paintings. As an advantageous and alternative methodology for the determination of egg, a new application of analytical pyrolysis, hyphenated with gas chromatography–mass spectrometry (GC–MS) system, in presence of hexamethyldisilazane (HMDS) and tetramethylammonium-hydroxide (TMAH), is reported here. The innovation lays mainly in the choice of new markers for the presence of egg. It is here demonstrated that in art diagnostic tris-TMS-ester and methyl ester of phosphoric acid, generated by the pyrolysis of standard phospholipids and synthetic painting layers containing egg as binding medium, may be used as new markers for identification of egg in tempera layers. The adoption of these new markers in analytical pyrolysis allows to obtain higher analytical performance with respect to classical markers (fatty acids), especially in terms of yield and, as a consequence, in terms of limit of detection.