Liberating Native Mass Spectrometry from Dependence on Volatile Salt Buffers by Use of Gábor Transform

Volatile salts, such as ammonium acetate, are commonly used in buffers for the analysis of intact proteins and protein complexes in native electrospray ionization mass spectrometry. Although these solutions are not technically buffers near pH 7, the volatile nature of the salt minimizes ion adduction to proteins upon transfer to vacuum. Conversely, common biochemical salt buffers, such as Tris/NaCl, are not traditionally used in native mass spectrometry because of the tendency of sodium and other ions to adduct to proteins or form large cluster ions, severely frustrating accurate mass assignment. Here, we demonstrate a Gábor transform method for extracting signal from native-like protein ions even in the presence of a large salt-cluster background. We further show the utility of this method in characterizing polymers and show that the measured average mass of long-chain polyethylene glycol ions from a commercial polymer sample is ∼30 % higher than the manufacturer-estimated average mass. It is expected that this method will enable more widespread use of conventional biochemical buffers in native mass spectrometry and decrease dependence on volatile salts.     

Dissecting heterogeneous molecular chaperone complexes using a mass spectrum deconvolution approach

Small heat-shock proteins (sHSPs) are molecular chaperones that prevent irreversible aggregation through binding nonnative target proteins. Due to their heterogeneity, these sHSP:target complexes remain poorly understood. We present a nanoelectrospray mass spectrometry analysis algorithm for estimating the distribution of stoichiometries comprising a polydisperse ensemble of oligomers. We thus elucidate the organization of complexes formed between sHSPs and different target proteins. We find that binding is mass dependent, with the resultant complexes reflecting the native quaternary architecture of the target, indicating that protection happens early in the denaturation. Our data therefore explain the apparent paradox of how variable complex morphologies result from the generic mechanism of protection afforded by sHSPs. Our approach is applicable to a range of polydisperse proteins and provides a means for the automated and accurate interpretation of mass spectra derived from heterogeneous protein assemblies.     

Methods for generating protein molecular ions in ToF-SIMS

One of the greatest challenges in mass spectrometry lies in the generation and detection of molecular ions that can be used to directly identify the protein from the molecular weight of the molecular ion. Typically, proteins are large (MW > 1000), nonvolatile, and/or thermally labile, but the vaporization process produced by many mass spectrometry techniques including time-of-flight secondary ion mass spectrometry (ToF-SIMS) is inherently limited to generating ions from smaller compounds or fragments of the parent molecule, making the identification of proteins complex. The application of specific molecules to aid in the generation of high molecular weight ions in ToF-SIMS has been recognized for some time. In this study we have developed a matrix-SAM substrate preparation technique based on the self-assembly of a matrix-like molecule, mercaptonicotinic acid (MNA), on gold. We then compare this substrate with two existing ToF-SIMS sample preparation techniques, cationized alkane thiol and matrix-enhanced SIMS (MESIMS). The results of this study illustrate that while there is a range of methods that can be used to improve the molecular ion yield of proteins in ToF-SIMS, their efficacy and reproducibility vary considerably and crucially are linked to the sample preparation and/or protein application methods used. Critically, the MNA modified substrate was able to simultaneously induce molecular ions for each protein present in a multicomponent solution, suggesting that this sample preparation technique may have future application in proteomics and DNA analysis.     

Native mass spectrometry—A valuable tool in structural biology

Proteins and the complexes they form with their ligands are the players of cellular action. Their function is directly linked with their structure making the structural analysis of protein‐ligand complexes essential. Classical techniques of structural biology include X‐ray crystallography, nuclear magnetic resonance spectroscopy and recently distinguished cryo‐electron microscopy. However, protein‐ligand complexes are often dynamic and heterogeneous and consequently challenging for these techniques. Alternative approaches are therefore needed and gained importance during the last decades. One alternative is native mass spectrometry, which is the analysis of intact protein complexes in the gas phase. To achieve this, sample preparation and instrument conditions have to be optimised. Native mass spectrometry then reveals stoichiometry, protein interactions and topology of protein assemblies. Advanced techniques such as ion mobility and high‐resolution mass spectrometry further add to the range of applications and deliver information on shape and microheterogeneity of the complexes. In this tutorial, we explain the basics of native mass spectrometry including sample requirements, instrument modifications and interpretation of native mass spectra. We further discuss the developments of native mass spectrometry and provide example spectra and applications.     

Metal displacement and stoichiometry of protein‐metal complexes under native conditions using capillary electrophoresis/mass spectrometry

Increases in the study of protein‐metal complexes, as well as in metal displacement in protein‐metal complexes under native conditions for optimum catalytic properties in drug research and catalyst design, demands a separation/detection technology that can accurately measure metal displacement and stoichiometry in protein‐metal complexes. Both nuclear magnetic resonance (NMR) and X‐ray diffraction techniques have been used for this purpose; however, these techniques lack sensitivity. Electrospray ionization mass spectrometry (ESI‐MS) using direct infusion offers higher sensitivity than the former techniques and provides molecular distribution of various protein‐metal complexes. However, since protein‐metal complexes under native conditions usually are dissolved in salt solutions, their direct ESI‐MS analysis requires off‐line sample clean‐up prior to MS analysis to avoid sample suppression during ESI. Moreover, direct infusion of the salty solution promotes non‐specific salt adduct formation by the protein‐metal complexes under ESI‐MS, which complicates the identification and stoichiometry measurements of the protein‐metal complexes. Because of the high mass of protein‐metal complexes and lack of sufficient resolution by most mass spectrometers to separate non‐specific from specific metal‐protein complexes, accurate protein‐metal stoichiometry measurements require some form of sample clean up prior to ESI‐MS analysis. In this study, we demonstrate that capillary electrophoresis/electrospray ionization in conjunction with a medium‐resolution (～10 000) mass spectrometer is an efficient and fast method for the measurement of the stoichiometry of the protein‐metal complexes under physiological conditions (pH ～7). The metal displacement of Co²⁺ to Cd²⁺, two metal ions necessary for activation in the monomeric AHL lactonase produced by B. thuringiensis, has been used as a proof of concept. Copyright © 2010 John Wiley & Sons, Ltd.     

Top-down characterization of proteins and drug-protein complexes using nanoelectrospray tandem mass spectrometry

We report a 'top-down' approach for characterization of proteins, and identification of binding sites in protein-drug complexes using nanoelectrospray ionization hybrid quadrupole time-of-flight tandem mass spectrometry (nanoESI-MS/MS). The efficiency of direct fragmentation of intact protein ions and the feasibility of this method were initially demonstrated using several well-characterized proteins with different molecular weights including metallothionein (6126 Da), cytochrome c (horse, 12360 Da), myoglobin (horse, 16592 Da), and hemoglobin (human, 64453 Da). Simply varying collision energy without enzyme digestion and gel or LC separation generated a range of peptide fragments of these proteins. Over 80% of these peptide ions matched those in the SWISS-PROT database with mass accuracy of 8 to 32 ppm with external calibration. This technique was further applied to fragment a cisplatin-metallothionein complex to identify the binding sites, demonstrating a potential application in the study of drug-protein binding.     

Novel UV assay for protein determination and the characterization of copper-protein complexes by mass spectrometry

A very simple, highly selective and sensitive assay of proteins based on the biuret absorption in the ultraviolet region has been developed. The well-known biuret assay is based on the reaction of proteins with copper ions under strong alkaline conditions to form a copper-protein complex. Yet, copper ions may seriously interfere with the determination if the measurement is made in the UV range. In the present approach, proteins mobilize copper ions from insoluble salts at different pH values, and the copper-protein complexes are investigated by UV spectrophotometry and mass spectrometry. Upon using copper phosphate, free copper ions do not interfere with the determination from 540 to 240 nm. Copper absorbance slowly increases from 240 to 190 nm where a blank with the reagents is recommended. A maximum absorbance for the copper-protein complex was found at 226 nm and high pH value. The stoichiometries of the copper-protein complexes measured directly with a mass spectrometer are pH dependent: half of the peptides without any histidine residue chelate just a single Cu²⁺ ion at pH 7.4 but each such peptide mobilizes from 1 to 6 Cu²⁺ ions at pH 10.3. To determine proteins, 1-1.5 ml of 1.8% KOH solution with 0-20 μg ml⁻¹ protein is treated with 25 mg of copper phosphate powder. The mixture is powerfully stirred, centrifuged, and the absorbance of the supernatant is measured at 226 nm in 1 cm quartz cuvettes against a blank of the reagents. The color system obeys Beer's law in the range 0.1-20 μg ml⁻¹ protein at this wavelength. The molar absorptivity value proved to be a characteristic of each protein being analyzed. Therefore, individual proteins should be used to plot calibration curves. This assay proved to be over 100 times more sensitive than the classical biuret procedure. The method is highly selective and the determination is little affected by the presence of other substances. All other important analytical parameters were studied and practical applicability of the method has been verified by the analysis of some biological materials.     

Mass spectrometric analysis of nanoscale sample volumes extracted from open microchannels after sample preconcentration applied on amyloid beta peptides

A new instrumental concept for extraction of nanovolumes from open microchannels (dimensions 150 μm?×?50 μm, length 10 mm) manufactured on silicon microchips has been used in combination with a previously developed method for preconcentrating proteins and peptides in the open channels through electromigration. The extracted nanovolumes were further analyzed using nanoelectrospray ionization (nESI) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) directly or with subsequent enzymatic protein digestion in a nanodroplet prior to the MS analysis. Preconcentration of the samples resulted in a 15-fold sensitivity increase in nESI for a neurotensin solution, and using MALDI-MS, amyloid beta (Aβ) peptides could be detected in concentrations down to 1 nM. The method was also successfully applied for detection of cell culture Aβ.     

Effects of Cations on Protein and Peptide Charging in Electrospray Ionization from Aqueous Solutions

The effects of eight different cations with ionic radii between 69 and 337 pm on the charging of peptides and proteins with electrospray ionization from aqueous acetate salt solutions are reported. Significant adduction occurs for all cations except NH₄ ⁺, and the average protein charge is lower when formed from solutions containing salts compared with solutions without salts added. Circular dichroism and ion mobility results show the protein conformations are different in pure water compared with salt solutions, which likely affects the extent of charging. The average charge of protein and peptide ions formed from solutions with Li⁺ and Cs⁺, which have Gibbs solvation free energies (GSFEs) that differ by 225 kJ/mol, is similar. Lower charge states are typically formed from solutions with tetramethylammonium and tetraethylammonium that have lower GSFE values. Loss of the larger cations that have the lowest GSFEs is facile when adducted protein ions are collisionally activated, resulting in the formation of lower analyte charge states. This reaction pathway provides a route to produce abundant singly protonated protein ions under native mass spectrometry conditions. The average protein and peptide charge with NH₄ ⁺ is nearly the same as that with Rb⁺ and K⁺, cations with similar GSFE and ionic radii. This indicates that proton transfer from NH₄ ⁺ to proteins plays an insignificant role in the extent of protein charging in native mass spectrometry.

A novel approach to analyze membrane proteins by laser mass spectrometry: From protein subunits to the integral complex

A novel laser-based mass spectrometry method termed LILBID (laser-induced liquid bead ion desorption) is applied to analyze large integral membrane protein complexes and their subunits. In this method the ions are IR-laser desorbed from aqueous microdroplets containing the hydrophobic protein complexes solubilized by detergent. The method is highly sensitive, very efficient in sample handling, relatively tolerant to various buffers, and detects the ions in narrow, mainly low-charge state distributions. The crucial experimental parameter determining whether the integral complex or its subunits are observed is the laser intensity: At very low intensity level corresponding to an ultrasoft desorption, the intact complexes, together with few detergent molecules, are transferred into vacuum. Under these conditions the oligomerization state of the complex (i.e., its quaternary structure) may be analyzed. At higher laser intensity, complexes are thermolyzed into subunits, with any residual detergent being stripped off to yield the true mass of the polypeptides. The model complexes studied are derived from the respiratory chain of the soil bacterium Paracoccus denitrificans and include complexes III (cytochrome bc(1) complex) and IV (cytochrome c oxidase). These are well characterized multi-subunit membrane proteins, with the individual hydrophobic subunits being composed of up to 12 transmembrane helices.     

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.     

Probing the hydrophobic effect of noncovalent complexes by mass spectrometry

The study of noncovalent interactions by mass spectrometry has become an active field of research in recent years. The role of the different noncovalent intermolecular forces is not yet fully understood since they tend to be modulated upon transfer into the gas phase. The hydrophobic effect, which plays a major role in protein folding, adhesion of lipid bilayers, etc., is absent in the gas phase. Here, noncovalent complexes with different types of interaction forces were investigated by mass spectrometry and compared with the complex present in solution. Creatine kinase (CK), glutathione S-transferase (GST), ribonuclease S (RNase S), and leucine zipper (LZ), which have dissociation constants in the nM range, were studied by native nanoelectrospray mass spectrometry (nanoESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking (XL). Complexes interacting with hydrogen bonds survived the transfer into gas phase intact and were observed by nanoESI-MS. Complexes that are bound largely by the hydrophobic effect in solution were not detected or only at very low intensity. Complexes with mixed polar and hydrophobic interactions were detected by nanoESI-MS, most likely due to the contribution from polar interactions. All noncovalent complexes could easily be studied by XL MALDI-MS, which demonstrates that the noncovalently bound complexes are conserved, and a real “snap-shot” of the situation in solution can be obtained.     

A study of Src SH2 domain protein-phosphopeptide binding interactions by electrospray ionization mass spectrometry

The noncovalent binding of various peptide ligands to pp60^src (Src) SH2 (Src homology 2) domain protein (12.9 ku) has been used as a model system for development of electrospray ionization mass spectrometry (ESI-MS) as a tool to study noncovalently bound complexes. SH2 motifs in proteins are critical in the signal transduction pathways of the tyrosine kinase growth factor receptors and recognize phosphotyrosine-containing proteins and peptides. ESI-MS with a magnetic sector instrument and array detection has been used to detect the protein-peptide complex with low-picomole sensitivity. The relative abundances of the multiply charged ions for the complex formed between Src SH2 protein and several nonphosphorylated and phosphorylated peptides have been compared. The mass spectrometry data correlate well to the measured binding constants derived from solution-based methods, indicating that the mass spectrometry-based method can be used to assess the affinity of such interactions. Solution-phase equilibrium constants may be determined by measuring the amount of bound and unbound species as a function of concentration for construction of a Scatchard graph. ESI-MS of a solution containing Src SH2 with a mixture of phosphopeptides showed the expected protein-phosphopeptide complex as the dominant species in the mass spectrum, demonstrating the method’s potential for screening mixtures from peptide libraries.     

Uncovering the Stoichiometry of Pyrococcus furiosus RNase P,a Multi‐Subunit Catalytic Ribonucleoprotein Complex,by Surface‐Induced Dissociation and Ion Mobility Mass Spectrometry

We demonstrate that surface‐induced dissociation (SID) coupled with ion mobility mass spectrometry (IM‐MS) is a powerful tool for determining the stoichiometry of a multi‐subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg²⁺. We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5′ maturation. Previous step‐wise, Mg²⁺‐dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21⋅RPP29 and POP5⋅RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21⋅RPP29 and (POP5⋅RPP30)₂ complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM‐MS in resolving conformational heterogeneity and yielding insights on RNP assembly.     

Uncovering the Stoichiometry of Pyrococcus furiosus RNase P,a Multi‐Subunit Catalytic Ribonucleoprotein Complex,by Surface‐Induced Dissociation and Ion Mobility Mass Spectrometry

We demonstrate that surface‐induced dissociation (SID) coupled with ion mobility mass spectrometry (IM‐MS) is a powerful tool for determining the stoichiometry of a multi‐subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg²⁺. We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5′ maturation. Previous step‐wise, Mg²⁺‐dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21?RPP29 and POP5?RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21?RPP29 and (POP5?RPP30)₂ complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM‐MS in resolving conformational heterogeneity and yielding insights on RNP assembly.     

Analysis of low‐density lipoprotein‐associated proteins using the method of digitized native protein mapping

The method of digitized native protein mapping, combining nondenaturing micro 2DE, grid gel‐cutting, and quantitative LC‐MS/MS (in data‐independent acquisition mode, or MS^E), was improved by using a new MS/MS mode, ion mobility separation enhanced‐MS^E (HDMS^E), and applied to analyze the area of human plasma low‐density lipoprotein (LDL). An 18 mm × 4.8 mm rectangular area which included LDL on a nondenaturing micro 2D gel of human plasma was grid‐cut into 72 square gel pieces and subjected to quantitative LC‐MS/MS. Compared with MS^E, HDMS^E showed significantly higher performance, by assigning 50% more proteins and detecting each protein in more squares. A total of 253 proteins were assigned with LC‐HDMS^E and the quantity distribution of each was reconstructed as a native protein map. The maps showed that Apo B‐100 was the most abundant protein in the grid‐cut area, concentrated at pI ca. 5.4–6.1 and apparent mass ca. 1000 kDa, which corresponded to four gel pieces, squares 39–42. An Excel macro was prepared to search protein maps which showed protein quantity peaks localized within this concentrated region of Apo B‐100. Twenty‐two proteins out of the 252 matched this criterion, in which 19 proteins have been reported to be associated with LDL. This method only requires several microliters of a plasma sample and the principle of the protein separation is totally different from the commonly used ultracentrifugation. The results obtained by this method would provide new insights on the structure and function of LDL.     

Affinity capture using chimeric membrane proteins bound to magnetic beads for rapid ligand screening by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

The rapid and specific detection of therapeutically important ligands in complex mixtures, that may bind to membrane proteins, remains challenging for many research laboratories and pharmaceutical industries. Through its use in the development of screening assays, mass spectrometry (MS) is currently experiencing a period of tremendous expansion. In the study presented here, we took advantage of the remarkable stability properties of a bacterial membrane protein, the KcsA K⁺ channel, produced in E. coli and purified as a tetrameric protein in the presence of a detergent. This membrane protein can subserve as a molecular template to display the pore‐forming region of human K⁺ channels, which are considered as targets in the search for inhibitory ligands. The engineered chimeric proteins were linked to metal‐bound magnetic beads, for the screening of complex peptide mixtures, such as that of scorpion venoms. The affinity‐captured scorpion toxins were eluted prior to matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS), and to nano‐electrospray ionization tandem mass QqTOF mass spectrometry (MS/MS) analysis. The de novo sequence of the toxins was deduced by combining the MS/MS fragmentation of the reduced form (up to the 33 first residues) and the trypsin digest peptides of the native toxins. This affinity‐capture screening assay led to the isolation and characterization of potent and specific ligands of the human K⁺ channel, Kv1.3. The affinity‐capture procedure is fast and reproducible. When linked to magnetic beads, the chimeric membrane protein can be re‐used several times without losing any of its selectivity or specificity. This assay also benefits from the fact that it requires minimal amounts of animal venoms or complex mixtures, which can be expensive or difficult to procure. Copyright © 2009 John Wiley & Sons, Ltd.     

Three-layer matrix/sample preparation method for MALDI MS analysis of low nanomolar protein samples

A robust and sensitive sample preparation method is presented for matrix-assisted laser desorption ionization (MALDI) mass spectrometric analysis of low nanomolar concentrations of proteins containing high amounts of common salts and buffers. This method involves the production of densely packed sub-micrometer matrix crystals by depositing a matrix solution on top of a matrix seed-layer prepared on a MALDI target. A sub-microliter aliquot of analyte solution is then directly added to the top of the matrix crystals to form a thin-layer. alpha-Cyano-4-hydroxycinnamic acid (4-HCCA) is used as matrix and demonstrated to give better performance than other commonly used matrices, such as 2,5-dihydroxybenzoic acid (DHB), 2-(4-hydroxy-phenylazo) benzoic acid (HABA), or sinapinic acid. This three-layer method is shown to be superior to the other MALDI sample preparation methods, particularly for handling low nanomolar protein solutions containing salts and buffers.