A generic strategy to analyze the spatial organization of multi-protein complexes by cross-linking and mass spectrometry

Most cellular functions are performed by multi-protein complexes. The identity of the members of such complexes can now be determined by mass spectrometry. Here we show that mass spectrometry can also be used in order to define the spatial organization of these complexes. In this approach, components of a protein complex are purified via molecular interactions using an affinity tagged member and the purified complex is then partially cross-linked. The products are separated by gel electrophoresis and their constituent components identified by mass spectrometry yielding nearest-neighbor relationships. In this study, a member of the yeast nuclear pore complex (Nup85p) was tagged and a six-member sub-complex of the pore was cross-linked and analyzed by 1D SDS-PAGE. Cross-linking reactions were optimized for yield and number of products. Analysis by MALDI mass spectrometry resulted in the identification of protein constituents in the cross-linked bands even at a level of a few hundred femtomoles. Based on these results, a model of the spatial organization of the complex was derived that was later supported by biological experiments. This work demonstrates, that the use of mass spectrometry is the method of choice for analyzing cross-linking experiments aiming on nearest neighbor relationships.     

Atmospheric pressure MALDI Fourier transform mass spectrometry of labile oligosaccharides

An atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) source coupled to Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS) under UV laser and solid matrix conditions has been demonstrated to analyze a variety of labile oligosaccharides including O-linked and N-linked complex glycans released from glycoproteins. Spectra were acquired by both AP MALDI and vacuum MALDI and directly compared. The results presented here confirm that AP MALDI can generate significantly less energetic ions than vacuum MALDI and is able to produce the intact molecular ions with little or no fragmentation in both positive and negative ion mode analyses. Under certain conditions, noncovalent complexes of sialylated oligosaccharides were observed. The sensitivity attainable by AP MALDI was found to be comparable to conventional MALDI, and tandem mass spectrometry of oligosaccharides ionized by AP MALDI was shown to allow detailed structural analysis. Analysis of N-glycan mixtures derived from human fibrinogen further demonstrated that AP MALDI-FT ICR MS is ideal for the study of complex glycan samples as it provides high-accuracy, high-resolution mass analysis with no difficulty in distinguishing sample constituents from fragment ions.     

Simultaneous mass analysis of positive and negative ions using a dual-polarity time-of-flight mass spectrometer

Positive and negative ions produced from matrix-assisted laser desorption/ionization (MALDI) were simultaneously measured using a newly developed dual-polarity time-of-flight mass spectrometer. This instrument is effective not only for express and comprehensive mass analysis but also for studying the ionization mechanisms of biomolecules. It comprises two identical time-of-flight mass analyzers located symmetrically about a MALDI ion source. The ion optics are arranged to be able to extract positive and negative ions synchronously with equal efficiency to each corresponding mass analyzer. Mass spectra of various proteins with molecular weights as large as that of myoglobin monomer and dimer were obtained. The spectral patterns obtained in this work are approximately mirror images with opposite polarities.     

Collisional cooling of large ions in electrospray mass spectrometry

Collisional cooling of ions in the rf-only multipole guides has become a method of choice for coupling electrospray sources to various mass analyzers. Normally parameters of such ion guides (length, pressure) provide enough thermalization and focusing for ions in a wide mass range. Noncovalent complexes, however, have more compact conformations than denatured biomolecules of similar mass and, therefore may not be transmitted efficiently through standard ion guides, as demonstrated by theoretical analysis, simulations, and experiments. Several methods of improving collisional cooling for large compact ions have been developed on a quadrupole time-of-flight instrument, which include operating the ion guides at higher pressure and trapping ions to increase the cooling time. Improved transmission of heavy ions obtained with those methods is studied in experiments with proteasome 20S, an oligomeric protein noncovalent complex with molecular weight around 692,000, and a few other compounds.     

Cation selectivity of natural and synthetic ionophores probed with laser-induced liquid beam mass spectrometry

The novel laser desorption method laser-induced liquid beam ionization/desorption (LILBID) is applied to the mass spectrometric examination of selective ion binding by natural and synthetic ionophores in methanol solutions. The ions are desorbed from a liquid jet with an IR laser pulse and then extracted perpendicularly into a reflectron time-of-flight (RE-TOF) analyzer. LILBID studies on the natural ion carriers valinomycin and monensin A are presented, as well as those on the synthetic crown ethers 18-crown-6, diaza-18-crown-6, and benzo-15-crown-5. No fragment ions are detected, and the measured ion selectivity is in good qualitative agreement with published stability constants of the complexes. The observed specific recognition of silver ions by diaza-18-crown-6 can be rationalized by the principle of hard and soft acids and bases, which predicts stable complexes when the polarizabilities of Lewis acid and base are similar. Weak, noncovalent interactions like those in the sandwich complex between two benzo-15-crown-5 molecules with one potassium ion are detected with LILBID. Their preservation during the process of ion desorption depends on the laser intensity. A comparison with spectra obtained by using electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) shows that LILBID can potentially become a sensitive tool for the screening of weak but specific molecular interactions.     

Ammonium dodecyl sulfate as an alternative to sodium dodecyl sulfate for protein sample preparation with improved performance in MALDI mass spectrometry

Sodium dodecyl sulfate (SDS) is a strong surfactant that is widely used in protein sample preparation. While protein and peptide samples containing up to approximately 1% SDS can be analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) using a two-layer matrix/sample deposition method, the presence of SDS in a protein sample generally degrades mass resolution and mass measurement accuracy. This degradation in performance is found to be related to the formation of sodium-protein adducts in the MALDI process. If the instrument resolving power is insufficient to separate these adduct peaks from the protonated molecular ion peak, peak broadening is observed in the protein molecular ion region, and as a result, the peak centroid shifts to a higher mass. In this work, we present a method using ammonium dodecyl sulfate as a viable alternative to SDS for protein sample preparation with much improved MALDI MS performance. Three non-sodium-based dodecyl sulfate surfactants, ammonium dodecyl sulfate (ADS), hydrogen dodecyl sulfate, and tris(hydroxymethyl)aminomethane dodecyl sulfate were investigated. Of the three surfactants tested, it is found that ADS gives the best performance in MALDI. For proteins with moderate molecular masses (i.e., up to approximately 25 kDa), the presence of ADS in a protein sample does not result in significant degradation in mass resolution and accuracy, and the protonated molecular ion peak is the dominant peak in the MALDI spectrum. The ammonium adduct ions dominate the MALDI spectra when the protein mass exceeds approximately 25 kDa; however, ADS still gives better results than SDS. The behavior of ADS in gel electrophoresis was also investigated. It is shown that cell extracts dissolved in ADS can be separated by normal SDS-polyacrylamide gel electrophoresis by simply mixing them with the SDS sample buffer. The application of ADS as the surfactant for protein solubilization with improved performance in MALDI analysis is demonstrated in the study of a detergent insoluble fraction from a Raji/CD9 B-cell lymphocyte lysate.     

Combining fluorescence detection and mass spectrometric analysis for comprehensive and quantitative analysis of redox-sensitive cysteines in native membrane proteins

Monobromobimane (MBB) is a lipophilic reagent that selectively modifies free cysteine residues in proteins. Because of its lipophilic character, MBB is capable of labeling cysteine residues in membrane proteins under native conditions. Reaction of MBB with the sulfhydryl groups of free cysteines leads to formation of highly fluorescent derivatives. Here we describe a procedure for the detection and relative quantitation of MBB-labeled cysteines using fluorescence and mass spectrometric analyses, which allow determination of free cysteine content and unambiguous identification of MBB-modified cysteine residues. We have applied this approach to the analysis of the free and redox-sensitive cysteine residues of a large membrane protein, the sarcoplasmic reticulum Ca2+ release channel with a molecular mass of 2.2 million Da. Labeling was performed under physiologic conditions where the channel complex is in its native environment and is functionally active. The purified MBB-labeled channel complex was enzymatically digested, and the resulting peptides were separated by reversed-phase high-performance chromatography. MBB-labeled peptides were detected by fluorescence and identified by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Under MALDI conditions, partial photolytic fragmentation of the MBB-peptide bound occurred, thus allowing convenient screening for the MBB-modified peptides in the MS spectrum by detection of the specific mass increment of 190.07 Da for MBB-modified cysteine residues. Modification of the peptides was further confirmed by tandem mass spectrometric analysis, utilizing sequencing information and the presence of the specific immonium ion for the MBB-modified cysteine residues at m/z 266.6. Quantitative information was obtained by comparison of both fluorescence and MS signal intensities of MBB-modified peptides. Combination of fluorescence with MS detection and analysis of MBB-labeled peptides supported by a customized software program provides a convenient method for identifying and quantifying redox-sensitive cysteines in membrane proteins of native biological systems. Identification of one redox-sensitive cysteine (2327) in the native membrane-bound sarcoplasmic reticulum Ca2+ release channel is described.     

High dynamic range bio-molecular ion microscopy with the Timepix detector

Highly parallel, active pixel detectors enable novel detection capabilities for large biomolecules in time-of-flight (TOF) based mass spectrometry imaging (MSI). In this work, a 512 × 512 pixel, bare Timepix assembly combined with chevron microchannel plates (MCP) captures time-resolved images of several m/z species in a single measurement. Mass-resolved ion images from Timepix measurements of peptide and protein standards demonstrate the capability to return both mass-spectral and localization information of biologically relevant analytes from matrix-assisted laser desorption ionization (MALDI) on a commercial ion microscope. The use of a MCP-Timepix assembly delivers an increased dynamic range of several orders of magnitude. The Timepix returns defined mass spectra already at subsaturation MCP gains, which prolongs the MCP lifetime and allows the gain to be optimized for image quality. The Timepix peak resolution is only limited by the resolution of the in-pixel measurement clock. Oligomers of the protein ubiquitin were measured up to 78 kDa.     

Determination of Block Length Distributions of Poly(oxypropylene) and Poly(oxyethylene) Block Copolymers by MALDI-FTICR Mass Spectrometry

Matrix-assisted laser desorption/ionization (MALDI) was performed on an external ion source Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) to analyze the block length distributions of triblock polymers of poly(oxypropylene) and poly(oxyethylene). The first series of results presented demonstrate that the apparent molecular weight distributions are distorted. This distortion is induced by the flight-time-induced mass discrimination inherent in the experimental technique, the variation of isotopic patterns over the measured mass range, and the overlap of peaks in the spectrum. Subsequently, a method for the treatment of molecular weight distributions measured by MALDI on an external ion source FTICR-MS is developed to yield the actual molecular weight distribution and, from that, the individual block length distributions. For the first time, detailed and accurate molecular weight data were obtained on a complex sample using this methodology, which independently validates the data provided by the manufacturer. The experimentally verified random coupling hypothesis proves the validity of the methodology.     

Unit mass baseline resolution for an intact 148 kDa therapeutic monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry

Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) provides the highest mass resolving power and mass measurement accuracy for unambiguous identification of biomolecules. Previously, the highest-mass protein for which FTICR unit mass resolution had been obtained was 115 kDa at 7 T. Here, we present baseline resolution for an intact 147.7 kDa monoclonal antibody (mAb), by prior dissociation of noncovalent adducts, optimization of detected total ion number, and optimization of ICR cell parameters to minimize space charge shifts, peak coalescence, and destructive ion cloud Coulombic interactions. The resultant long ICR transient lifetime (as high as 20 s) results in magnitude-mode mass resolving power of ~420,000 at m/z 2,593 for the 57+ charge state (the highest mass for which baseline unit mass resolution has been achieved), auguring for future characterization of even larger intact proteins and protein complexes by FTICR MS. We also demonstrate up to 80% higher resolving power by phase correction to yield an absorption-mode mass spectrum.     

Anti-viral inhibitor binding to influenza neuraminidase by MALDI mass spectrometry

A matrix-assisted laser desorption ionization (MALDI) mass spectrometry-based approach is applied to identify active site domains within influenza neuraminidase that bind the antiviral inhibitors zanamivir (ZANA) and 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA). Combined data from the tryptic and Glu-C endoproteinase digests of neuraminidase-inhibitor complexes have identified binding peptides that contain the active site residues Arg118, Glu119, Arg156, Glu276, and Tyr406. The binding of these residues was confirmed from the analysis of available X-ray crystal structures. The ability to identify peptides within the active sites of proteins and likely binding residues provides both a rapid and relatively high throughput approach with which to screen protein-drug interactions by MALDI mass spectrometry.     

Metal-enhanced fluorescence of chlorophylls in single light-harvesting complexes

Ensemble and single-molecule spectroscopy demonstrates that both emission and absorption of peridinin-chlorophyll-protein photosynthetic antennae can be largely enhanced through plasmonic interactions. We find up to 18-fold increase of the chlorophyll fluorescence for complexes placed near a silver metal layer. This enhancement, which leaves no measurable effects on the protein structure, is observed when exciting either chlorophyll or carotenoid and is attributed predominantly to an increase of the excitation rate in the antenna. The enhancement mechanism comes from plasmon-induced amplification of electromagnetic fields inside the complex. This result is an important step toward applying plasmonic nanostructures for controlling the optical response of complex biomolecules and improving the design and functioning of artificial light-harvesting systems.     

Solvent-free MALDI-MS for the analysis of a membrane protein via the mini ball mill approach: case study of bacteriorhodopsin

A mini ball mill (MBM) solvent-free matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) method allows for the analysis of bacteriorhodopsin (BR), an integral membrane protein that previously presented special analytical problems. For well-defined signals in the molecular ion region of the analytes, a desalting procedure of the MBM sample directly on the MALDI target plate was used to reduce adduction by sodium and other cations that are normally attendant with hydrophobic peptides and proteins as a result of the sample preparation procedure. Mass analysis of the intact hydrophobic protein and the few hydrophobic and hydrophilic tryptic peptides available in the digest is demonstrated with this robust new approach. MS and MS/MS spectra of BR tryptic peptides and intact protein were generally superior to the traditional solvent-based method using the desalted "dry" MALDI preparation procedure. The solvent-free method expands the range of peptides that can be effectively analyzed by MALDI-MS to those that are hydrophobic and solubility-limited.     

Effect of local matrix crystal variations in matrix-assisted ionization techniques for mass spectrometry

Intense intact molecular ion signals have been obtained from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidyiinositol using matrix-enhanced secondary ion mass spectrometry (ME-SIMS). It was found that the high-mass (m/z >500) regions of the ME-SIMS spectra closely resembled those obtained using matrix-assisted laser desorption/ionization (MALDI). Using high spatial resolution SIMS, a detailed investigation of dried-droplet samples was performed. Based on the detected Na+ and 2,5-DHB matrix signal intensities, different crystal types were distinguished, in addition to different sizes of crystals. Spatially mapping the pseudomolecular and fragment ions of the phospholipids revealed that the nature of the pseudomolecular ions formed, as well as the ratio of intact molecular to fragment ion, was dependent on the type and surface composition of the crystal. The observed chemical bias effects due to crystal heterogeneity and the resulting variation in desorption/ionization efficiency will complicate the interpretation of data obtained from matrix-assisted mass spectrometric (imaging) techniques and is an important factor in the "hot spot" phenomenon frequently encountered in MALDI experiments. In this respect, imaging SIMS was found to be a versatile tool to investigate the effects of the local physicochemical conditions on the detected molecular species.     

Probing the effects of cone potential in the electrospray ion source: consequences for the determination of molecular weight distributions of synthetic polymers

Shifts in the relative intensities of oligomer ions are found to accompany changes in the cone potential in the electrospray ion source, which introduce uncertainties into average molecular weight determinations for polymer distributions. Similar shifts with changes in cone potential have long been recognized in the multiple-charge distributions of proteins and other biomolecules. In the case of multiple-charge distributions of a single, or small number of, species there are no major consequences for calculation of molecular weight; however, mass distributions and the averages thereof, are of major concern with synthetic polymers and understanding the shifts in relative intensities becomes critically important. We report here an evaluation of the effects of cone potentials on the molecular weight distributions of synthetic polymers, which we compare with the effects on charge-state distributions of peptides. The effects of cone potential have been modeled mathematically, from which we conclude that cone potentials exert a focusing effect dependent on the mass-to-charge ratios of ions. It is largely this focusing effect that determines the dependence of oligomer ion intensities upon cone potential in the ESI mass spectra of polymers. The influence of cone potential on molecular weight determinations of polymers of varying polydispersities (P(o)) is compared and discussed. For polymers with low polydispersities (e.g., narrow molecular weight poly(ethyleneglycol) standards with P(o) < 1.5), the variation in molecular weight determinations tends to be small (typically <5%), whereas with synthetic polymers with polydispersities greater than 2, variations in cone potential can influence molecular weight determinations significantly (by 100% or even more).     

MALDI ion trap mass spectrometer with charge detector for large biomolecule detection

Up to now, all commercial matrix-assisted laser desorption/ionization (MALDI) mass spectrometers still can not efficiently analyze very large biomolecules. In this work, we report the development of a novel MALDI ion trap mass spectrometer which can enrich biomolecular ions to enhance the detection sensitivity. A charge detector was installed to measure the large ions directly. With this design, we report the first measurement of IgM with the mass-to-charge ratio (m/z) at 980?000. In addition, quantitative measurements of the number of ions can be obtained. A step function frequency scan was first developed to get a clear signal in the m/z range from 200,000 to 1,000,000.     

Immunoassays with direct mass spectrometric detection

A rapid, specific, and sensitive method for the detection of protein-protein interactions is of crucial importance for drug discovery and clinical diagnostics. Mass spectrometry plays a major role in the analysis of proteins, but its application to the routine analysis of protein complexes has been lagging behind. A new strategy for high-throughput analysis of protein interactions is presented here. We demonstrate application to immunochemical questions such as epitope mapping, kinetic studies, and sandwich assays. The methodology is based on a direct mass spectrometric readout for antigen-antibody complexes in the 150-400 kDa range. This has become possible using a novel detector technology and chemical cross-linking to stabilize complexes for analysis by MALDI MS. We demonstrate high detection sensitivity (femtomole quantities of antigen), high specificity (specific detection of antigen directly in serum), high accuracy, and high speed (minutes per assay), surpassing conventional analytical methods by more than 2 orders of magnitude.     

Detection of phosphopeptides using Fe(III)-nitrilotriacetate complexes immobilized on a MALDI plate

Metal affinity complexes were chemically grafted onto the surface of gold matrix-assisted laser desorption/ionization (MALDI) plates by coupling a derivative of nitrilotriacetate (NTA) to immobilized poly(acrylic acid) (PAA) and subsequently forming the Fe(III)-NTA complex. The immobilized complexes can adsorb phosphorylated peptides preferentially from protein digests; deposition of digests on these surface-modified plates, followed by rinsing with an acetic acid solution, addition of matrix, and subsequent analysis by MALDI MS, resulted in mass spectra dominated by peaks corresponding to phosphopeptides. In the case of analyzing a tryptic digest of beta-casein, conventional MALDI MS revealed only one monophosphopeptide, while use of the Fe(III)-NTA-PAA-modified plate resulted in strong signals due to two additional tetraphosphorylated species. The diminution or elimination of signals due to nonphosphorylated species also greatly simplified the identification of phosphopeptides during analysis of ovalbumin digests and myoglobin digests spiked with an equimolar mixture of angiotensin and phosphoangiotensin. The matrix 2',4',6'-trihydroxyacetophenone mixed with diammonium hydrogen citrate proved to be much better than alpha-cyano-4-hydroxycinnamic acid for the detection of phosphorylated peptides from digests of beta-casein and ovalbumin.     

Robust data processing and normalization strategy for MALDI mass spectrometric imaging

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) provides localized information about the molecular content of a tissue sample. To derive reliable conclusions from MSI data, it is necessary to implement appropriate processing steps in order to compare peak intensities across the different pixels comprising the image. Here, we review commonly used normalization methods, and propose a rational data processing strategy, for robust evaluation and modeling of MSI data. The approach includes newly developed heuristic methods for selecting biologically relevant peaks and pixels to reduce the size of a data set and remove the influence of the applied MALDI matrix. The methods are demonstrated on a MALDI MSI data set of a sagittal section of rat brain (4750 bins, m/z = 50-1000, 111 × 185 pixels) and the proposed preferred normalization method uses the median intensity of selected peaks, which were determined to be independent of the MALDI matrix. This was found to effectively compensate for a range of known limitations associated with the MALDI process and irregularities in MS image sampling routines. This new approach is relevant for processing of all MALDI MSI data sets, and thus likely to have impact in biomarker profiling, preclinical drug distribution studies, and studies addressing underlying molecular mechanisms of tissue pathology.     

Atmospheric pressure molecular imaging by infrared MALDI mass spectrometry

An atmospheric pressure (AP) MALDI imaging interface was developed for an orthogonal acceleration time-of-flight mass spectrometer and utilized to analyze peptides, carbohydrates, and other small biomolecules using infrared laser excitation. In molecular imaging experiments, the spatial distribution of mock peptide patterns was recovered with a detection limit of approximately 1 fmol/pixel from a variety of MALDI matrixes. With the use of oversampling for the image acquisition, a spatial resolution of 40 microm, 5 times smaller than the laser spot size, was achieved. This approach, however, required that the analyte was largely removed at the point of analysis before the next point was interrogated. Native water in plant tissue was demonstrated to be an efficient natural matrix for AP infrared laser desorption ionization. In soft fruit tissues from bananas, grapes, and strawberries, potassiated ions of the most abundant metabolites, small carbohydrates, and their clusters produced the strongest peaks in the spectra. Molecular imaging of a strawberry skin sample revealed the distribution of the sucrose, glucose/fructose, and citric acid species around the embedded seeds. Infrared AP MALDI mass spectrometric imaging without the addition of an artificial matrix enables the in vivo investigation of small biomolecules and biological processes (e.g., metabolomics) in their natural environment.