Multiply Charged Protein Ions Research Articles

Detection of multiply charged protein ions using matrix-assisted laser desorption/ionization mass spectrometry and a force-dried droplet method with a 2-nitrophloroglucinol matrix.

Conventional dried droplet (DD) methods show poor reproducibility in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) due to the frequent induction of a heterogeneous sample distribution. Recently, a forced dried droplet (FDD) sample preparation method was introduced to form homogeneous samples; this method improves the reproducibility of MALDI-MS analysis and generates highly multiply charged ions compared to DD methods. The FDD method utilizes secondary nucleation to generate a homogeneous sample distribution by applying an external force such as fluid shear stress by stirring the sample using a micropipette tip. In this study, a 2-nitrophloroglucinol (2-NPG) matrix was used for the DD and FDD sample preparation methods, and the charge state and homogeneity were compared by detecting multiply charged ions of proteins including cytochrome c, myoglobin, and immunoglobulin G (IgG) and measuring the relative standard deviation (RSD). FDD with a 2-NPG matrix produced a more homogeneous sample distribution and higher charge state ions than the DD method. FDD with a 2-NPG matrix was applied in MALDI-MS analysis of IgG fragments obtained from sequential reduction of IgG. In addition, FDD with intentional scratching of the MALDI plate by rotating a micropipette tip was found to provide similar or better reproducibility, higher charge state ions, and more uniformly distributed sample morphology compared to FDD without scratching.

Generation of multiply charged ions from homogeneous MALDI microcrystals

The homogeneity of matrix assisted laser desorption/ionization (MALDI) microcrystals, laser fluence, and the proton affinity (PA) of matrices determine the generation of multiply charged ions. Here we adopt three structurally similar matrices, i.e. α-cyano-4-hydroxycinnamic acid (CHCA), 4-chloro-α-cyanocinnamic acid (Cl-CCA), and (E)-propyl α-cyano-4-hydroxyl cinnamylate (CHCA-C3) to examine the homogeneity of microcrystals by measuring the relative standard deviation (RSD) values of the matrices with the homogeneous sample preparation method, e.g. forced dried droplet (FDD). The average RSD values of CHCA, Cl-CCA, and CHCA-C3 matrices are ∼15%, ∼10%, and ∼20%, respectively. With the same CHCA matrix, we found the charge state of myoglobin ions can obtain more charges (+6) with the FDD method as compared to +5 charges obtained by the inhomogeneous sample preparation method, i.e. dried droplet (DD). The laser fluence also affects the generation of multiply charged ions. For instance, the maximum charge states of myoglobin and cytochrome c ions are +6 with CHCA and +4 with CHCA-C3 at threshold laser fluence and decreases at elevated laser fluence. The charge state of myoglobin ions with Cl-CCA matrix is +3 at threshold laser fluence and increases to the maximum charge state of +6 and eventually reduces to +5 with elevated laser fluence. Moreover, we found low PA value of matrix results in high charge state of analyte ions and vice versa. The matrix PA modulates the proton transfer reaction, leading to the formation of multiply charged ions. The homogeneous samples help control the charge states of ions and generate the multiply charged protein ions which can be used as potential mass calibration standards to search suitable protein samples at high m/z ratios with a MALDI time-of-flight mass spectrometer.

Time-Resolved Pulsed Hydrogen/Deuterium Exchange Mass Spectrometry Probes Gaseous Proteins Structural Kinetics

A pulsed hydrogen/deuterium exchange (HDX) method has been developed for rapid monitoring of the exchange kinetics of protein ions with D2O a few milliseconds after electrospray ionization (ESI). The stepwise gradual evolution of HDX of multiply charged protein ions was monitored using the pulsed HDX mass spectrometry technique. Upon introducing a very short pulse of D2O (in the μs to ms time scale) into the linear ion trap (LIT) of a time-of-flight (TOF) mass spectrometer, bimodal distributions were detected for the ions of cytochrome c and ubiquitin. Mechanistic details of HDX reactions for ubiquitin and cytochrome c in the gas phase were uncovered and the structural transitions were followed by analyzing the kinetics of HDX.

Development of a Human IgG4 Bispecific Antibody for Dual Targeting of Interleukin-4 (IL-4) and Interleukin-13 (IL-13) Cytokines

Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes.

Optimization in Detecting Multiply-charged Protein Ions using MALDI TOF-MS

The effects of trifluoroacetic acid (TFA) were evaluated on the generation of multiply charged ions of cytochrome c in a 2-nitrophloroglucinol (2-NPG) matrix in high-vacuum, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The presence of 1% TFA in the 2-NPG matrix solution was more effective in generating multiply charged protein ions than matrix solutions containing 0.1% or 0% TFA. Regarding the matrix itself, with 1% TFA, 2-NPG was significantly more effective in generating multiply charged ions than 2,5-dihydroxybenzoic acid (2,5-DHB). The maximum charge state of cytochrome c was +8 when using a 2-NPG matrix containing 1% TFA.

Identification of multiply charged proteins and amino acid clusters by liquid nitrogen assisted spray ionization mass spectrometry

The development of liquid nitrogen assisted spray ionization mass spectrometry (LNASI MS) for the analysis of multiply charged proteins (insulin, ubiquitin, cytochrome c, α-lactalbumin, myoglobin and BSA), peptides (glutathione, HW6, angiotensin-II and valinomycin) and amino acid (arginine) clusters is described. The charged droplets are formed by liquid nitrogen assisted sample spray through a stainless steel nebulizer and transported into mass analyzer for the identification of multiply charged protein ions. The effects of acids and modifier volumes for the efficient ionization of the above analytes in LNASI MS were carefully investigated. Multiply charged proteins and amino acid clusters were effectively identified by LNASI MS. The present approach can effectively detect the multiply charged states of cytochrome c at 400nM. A comparison between LNASI and ESI, CSI, SSI and V-EASI methods on instrumental conditions, applied temperature and observed charge states for the multiply charged proteins, shows that the LNASI method produces the good quality spectra of amino acid clusters at ambient conditions without applied any electric field and heat. To date, we believe that the LNASI method is the most simple, low cost and provided an alternative paradigm for production of multiply charged ions by LNASI MS, just as ESI-like ions yet no need for applying any electrical field and it could be operated at low temperature for generation of highly charged protein/peptide ions.

Alkali Metal Cation-Induced Destabilization of Gas-Phase Protein–Ligand Complexes: Consequences and Prevention

Electrospray ionization, now a well established technique for studying noncovalent protein-ligand interactions, is prone to production of alkali metal adducts. Here it is shown that this adduction significantly destabilizes the interactions between two model proteins and their ligands and that destabilization correlates with cation size. For both the [FKBP·FK506] and [lysozyme·NAG(n)] systems, dissociation of the metalated complex occurs at markedly lower collision energies than their purely protonated equivalents. Dependency upon size of the metal(+) demonstrates the importance of electrostatic charge density during the dissociation process. Differences in the gas phase basicities (GBapp) of the multiply charged protein ions and proton and sodium affinities of the ligands explain the observed charge partitioning during dissociation of the complexes. Ion mobility-mass spectrometry measurements demonstrate that metal cation adduction does not induce a significant increase in unfolding of the polypeptides, indicating that this is not the principal mechanism responsible for destabilization. Destabilizing effects can be largely reduced by exposing the electrospray to solvent (e.g., acetonitrile) vapor, a method that acts to reduce the amount of adduct formation as well as decrease the charge states of the resulting ions. This approach leads to more accurate determination of apparent K(D)s in the presence of trace alkali metals.

Controlling Hydrogen Scrambling in Multiply Charged Protein Ions during Collisional Activation: Implications for Top-Down Hydrogen/Deuterium Exchange MS Utilizing Collisional Activation in the Gas Phase

Hydrogen exchange in solution combined with ion fragmentation in the gas phase followed by MS detection emerged in recent years as a powerful tool to study higher order protein structure and dynamics. However, a certain type of ion chemistry in the gas phase, namely, internal rearrangement of labile hydrogen atoms (the so-called hydrogen scrambling), is often cited as a factor limiting the utility of this experimental technique. Although several studies have been carried out to elucidate the roles played by various factors in the occurrence and the extent of hydrogen scrambling, there is still no consensus as to what experimental protocol should be followed to avoid or minimize it. In this study we employ fragmentation of mass-selected subpopulations of protein ions to assess the extent of internal proton mobility prior to dissociation. A unique advantage of tandem MS is that it not only provides a means to map the deuterium content of protein ions whose overall levels of isotope incorporation can be precisely defined by controlling the mass selection window, but also correlates this spatial isotope distribution with such global characteristic as the protein ion charge state. Hydrogen scrambling does not occur when the charge state of the precursor protein ions selected for fragmentation is high. Fragment ions derived from both N- and C-terminal parts of the protein are equally unaffected by scrambling. However, spatial distribution of deuterium atoms obtained by fragmenting low-charge-density protein ions is consistent with a very high degree of scrambling prior to the dissociation events. The extent of hydrogen scrambling is also high when multistage fragmentation is used to probe deuterium incorporation locally. Taken together, the experimental results provide a coherent picture of intramolecular processes occurring prior to the dissociation event and provide guidance for the design of experiments whose outcome is unaffected by hydrogen scrambling.

Investigation of Deprotonation Reactions on Globular and Denatured Proteins at Atmospheric Pressure by ESSI-MS

Deprotonation reactions of multiply charged protein ions have been studied by introducing volatile reference bases at atmospheric pressure between an electrosonic spray ionization (ESSI) source and the inlet of a mass spectrometer. Apparent gas-phase basicities (GB(app)) of different charge states of protein ions were determined by a bracketing approach. The results obtained depend on the conformation of the protein ions in the gas phase, which is linked to the type of buffer used (denaturing or nondenaturing). In nondenaturing buffer, the GB(app) values are consistent with values predicted by the group of Kebarle using an electrostatic model (J. Mass Spectrom.2002, 38, 618) based on the crystal structures, but taking into account salt bridges between ionized basic and acidic sites on the protein surface. A new basicity order for the most basic sites was therefore obtained. An excellent agreement with the charge residue model (CRM) is obtained when comparing the observed and calculated maximum charge state. Decharging of the proteins in the electrosonic spray process could be also useful in the study on noncovalent complexes, by decreasing repulsive electrostatic interactions. A unified mechanism of the ESSI process is proposed.

Transmission Mode Ion/Ion Proton Transfer Reactions in a Linear Ion Trap

A new method is described for effecting ion/ion proton transfer reactions that involves storage of analyte ions while oppositely charged ions are transmitted through the stored ion population. In this approach, the products are captured and stored in the linear ion trap for subsequent mass analysis. Charge reduction of multiply charged protein ions is used as an example to illustrate the analytical usefulness of this method. In another variation of the transmission mode ion/ion reaction approach, two charge inversion experiments, implemented by passing analyte ions through a population of multiply charged reagent ions in a LIT, are also demonstrated. A pulsed dual ion source approach coupled with a hybrid triple quadrupole/linear ion trap instrument was used to demonstrate these two methods. The results for ion/ion reactions implemented using these so-called "transmission mode" experiments were comparable to those acquired using the more conventional mutual storage mode, both in terms of efficiency and information content of the spectra. An advantage of transmission mode experiments compared with mutual storage mode experiments is that they do not require any specialized measures to be taken to enable the simultaneous storage of oppositely charged ions.

Frequency Scan of a Quadrupole Mass Analyzer in the Third Stability Region for Protein Analysis

AbstractA linear quadrupole mass spectrometer operating in the third stability region [(a, q) ≈ (3, 3)] has been developed and utilized to obtain the mass spectra of multiply charged protein ions generated by electro‐spray ionization. The spectra were acquired by scanning the frequency of the quadrupole with a home‐built wide‐band power amplifier from 400 to 100 kHz at constant voltage amplitudes in the range of 100 V. A mass resolution approaching R1/2 = 200 was achieved for doubly charged gramicidin S ions at m/z 570.5. Further improvement of the resolution is anticipated when a higher‐voltage power amplifier is available.

Pulsed Dual Electrospray Ionization for Ion/Ion Reactions

A pulsed dual electrospray ionization source has been developed to generate positive and negative ions for subsequent ion/ion reaction experiments. The two sprayers, typically a nano-electrospray emitter for analytes and an electrospray emitter for reagents, are positioned in a parallel fashion close to the sampling orifice of a triple quadrupole/linear ion trap tandem mass spectrometer (Sciex Q TRAP). The potentials applied to each sprayer are alternately pulsed so that ions of opposite polarity are generated separately in time. Ion/ion reactions take place after ions of each polarity are sequentially injected into a high-pressure linear ion trap, where axial trapping is effected by applying an auxiliary radio frequency voltage to the end lenses. The pulsed dual electrospray source allows optimization of each sprayer and can be readily coupled to any spray interface with no need for instrument modifications, provided the potentials required to transmit the ion polarity of interest can be alternated in synchrony with the emitter potentials. Ion/ion reaction examples such as charge reduction of multiply charged protein ions, charge inversion of peptides ions, and protein-protein complex formation are given to illustrate capabilities of the pulsed dual electrospray source in the study of gas-phase ion/ion chemistry.

Preparative Soft and Reactive Landing of Multiply Charged Protein Ions on a Plasma-Treated Metal Surface

Soft landing on a plasma-treated metal surface of multiply protonated protein ions from the gas phase results in a substantial retention of protein function, as demonstrated for trypsin and streptavidin. The majority of trypsin ions soft-landed at hyperthermal kinetic energies are undamaged and retain 72-98% of enzymatic activity after being washed into solution. A small fraction of trypsin ions that were landed at nominal kinetic energies of 130-200 eV remain tethered to the surface and show approximately 50% enzymatic activity. The streptavidin tetramer is found to dissociate to monomer units upon multiple charging in electrospray. The majority of soft-landed monomers can be washed into solution where they show affinity to biotin. The layer of streptavidin monomer that is immobilized on the surface can be detected if fluorescence-tagged and retains the ability to reversibly bind biotin. A mechanism is proposed to explain nondestructive protein ion discharge on the surface that considers proton migration from the soft-landed cations to the metal oxide layer and metal ion reduction by electron transfer from the bulk metal.

Electrosonic spray ionization. A gentle technique for generating folded proteins and protein complexes in the gas phase and for studying ion-molecule reactions at atmospheric pressure.

Electrosonic spray ionization (ESSI), a variant on electrospray ionization (ESI), employs a traditional micro ESI source with supersonic nebulizing gas. The high linear velocity of the nebulizing gas provides efficient pneumatic spraying of the charged liquid sample. The variable electrostatic potential can be tuned to allow efficient and gentle ionization. This ionization method is successfully applied to aqueous solutions of various proteins at neutral pH, and its performance is compared to that of the nanospray and micro ESI techniques. Evidence for efficient desolvation during ESSI is provided by the fact that the peak widths for various multiply charged protein ions are an order of magnitude narrower than those for nanospray. Narrow charge-state distributions compared to other ESI techniques are observed also; for most of the proteins studied, more than 90% of the protein ions can be accumulated in one charge state using ESSI when optimizing conditions. The fact that the abundant charge state is normally as low or lower than that recorded by ESI or nanospray indicates that folded protein ions are generated. The sensitivity of the ionization technique to high salt concentrations is comparable to that of nanospray, but ESSI is considerably less sensitive to high concentrations of organic additives such as glycerol or 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris base). Noncovalent complexes are observed in the case of myoglobin, protein kinase A/ATP complex, and other proteins. The extent of dissociation of protein ions in ESSI is comparable to or even smaller than that in the case of nanospray, emphasizing the gentle nature of the method. The unique features of ESSI are ascribed to very efficient spraying and the low internal energy supplied to the ions. Evidence is provided that the method is capable of generating fully desolvated protein ions at atmospheric pressure. This allows the technique to be used for the study of ion-molecule reactions at atmospheric pressure and examples of this are shown.

Studies into the selective accumulation of multiply charged protein ions in a quadrupole ion trap mass spectrometer

In seeking to improve the limit of detection of an electrospray ionization/quadrupole ion trap mass spectrometer (ESI/ITMS) for the analysis of multiply charged protein ions, studies related to the selective accumulation of these ions during ion injection have been undertaken. This was achieved by the interfacing of an Odyssey data system and the associated electronics used for arbitrary waveform generation from a Fourier transform mass spectrometer (FTMS) (ThermoFinnigan, Bremen, Germany) to an LCQ™ ‘classic’ quadrupole ion trap mass spectrometer (ThermoFinnigan, San Jose, CA). This allows the application of user defined stored waveform inverse Fourier transform (SWIFT) waveforms. These studies examined the efficiency of ejection and isolation at different q z values for a particular charge state from the distribution of ions usually observed with the electrospray ionization of proteins, and the variation in the intensity of a particular ion as a function of the accumulation time. In addition, this selective accumulation technique allowed for an order of magnitude improvement in the detection limit of this instrument, with the resulting reduction in space charge producing a five-fold increase in spectral resolution (FWHM) under the conditions studied.

Generating multiply charged protein ions by ultrasonic nebulization/multiple channel-electrospray ionization mass spectrometry.

An ultrasonic nebulization/multiple channel electrospray ionization (USN/MC-ES) source, which generates multiply charged peptides and proteins ions, was developed. The source is an ultrasonic nebulizer that is connected to a multiple channel electrospray ionization source. Aerosols were formed by ultrasonically nebulizing the sample solution. The aerosols were then purged into the central channel of a seven-channel ES source via nitrogen gas. A methanol solution that contained 1% trifluroacetic acid was electrosprayed through the outlying six electrosprayers. Detection of multiply charged peptide and protein ions indicated that electrospray was generated from the charged droplet containing analyte. The sample aerosol appeared to fuse with the charged methanol droplet in the air. Then electrospray ionization of the analyte occurred from the newly formed droplet. The peptide and protein prepared in deionized water were detected by this USN/MC-ES-MS. By varying the electrospray solvents, the signals of certain components in the mixture were selectively suppressed.

Identification of bacteriophage MS2 coat protein from E. coli lysates via ion trap collisional activation of intact protein ions.

Collisional activation of the intact MS2 viral capsid protein with subsequent ion/ion reactions has been used to identify the presence of this virus in E. coli lysates. Tandem ion trap mass spectrometry experiments on the +7, +8, and +9 charge states, followed by ion/ion reactions, provided the necessary sequence tag information (and molecular weight data) needed for protein identification via database searching. The most directly informative structural information is obtained from those charge states that produce a series of product ions arising from fragmentation at adjacent residues. The formation of these product ions via dissociation at adjacent amino acid residues depends greatly on the charge state of the parent ion. Database searching of the charge-state-specific sequence tags was performed by two different search engines: the ProteinInfo program from the Protein information Retrieval On-line World Wide Web Lab or PROWL and the TagIdent program from the ExPASy molecular biology server. These search engines were used in conjunction with the sequence tag information generated via collisional activation of the intact viral coat protein. These programs were used to evaluate the feasibility of generating sequence tags from collisional activation of intact multiply charged protein ions in a quadrupole ion trap.

Real-time monitoring of the gas phase reactions of a single ion population using the remeasurement experiment in Fourier transform ion cyclotron resonance mass spectrometry.

A single population of multiply charged protein ions formed by electrospray ionization is held in a trapped ion cell and remeasured continuously by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry while undergoing multiple reactions with diethylamine. In a first example, electrosprayed horse myoglobin with an average of 16 attached protons is reacted with base at a pressure of 1.5 x 10(-8) Torr for a period of 60 s. A total of 31 spectra acquired with a duty cycle of 2 s exhibit the charge state-dependent formation of up to three diethylamine adducts and removal of up to nine protons. A realtime measuring experiment is then conducted over a 1 h period to observe charge stripping of electrosprayed myoglobin ions, leaving as few as seven charges. Realtime monitoring is used to evaluate the effect of reagent gas pressure on adduct formation and is used in conjunction with high mass resolution FTICR detection to resolve the isotopic peaks within individual charge states of adduct spectra. The attractive features of real-time reaction monitoring include a dramatic reduction in experiment time and sample consumed while concurrently observing long-lived intermediates and reaction products as they form.

Remeasurement of electrosprayed proteins in the trapped ion cell of a Fourier transform ion cyclotron resonance mass spectrometer

A single population of multiply charged protein ions formed by electrospray ionization (ESI) is subjected to multiple Fourier transform ion cyclotron resonance (FTICR) excitation and detection events without promoting ion loss from the trapped ion cell. This nondestructive approach to mass spectrometric detection will allow detection limits to be significantly reduced by averaging the repetitive time-domain response that follows each excitation event. Under appropriate conditions, unit remeasurement efficiency is observed for large proteins; for example, 250 consecutive remeasurements of a single population of bovine albumin dimer ions (MW 132,532) yield the expected 16-fold improvement in signal-to-noise ratio compared to a single measurement. Examples presented include a 14-fmol sample of horse myoglobin (MW 16,951), which yields a 15:1 S/N for 50 remeasurements compared to a single scan S/N of 2:1 and a 30-fmol sample of bovine albumin dimer which exhibits a S/N of 25:1 for 50 remeasurements compared to a single scan S/N of 3:1. Of both practical and theoretical interest is the observation of a rapid collision-mediated homogeneous relaxation process that can return these large ions to the center of the cell within a few hundred milliseconds after excitation.