Multidimensional nano-HPLC coupled with tandem mass spectrometry for analyzing biotinylated proteins

Multidimensional high-performance liquid chromatography (HPLC) is a key method in shotgun proteomics approaches for analyzing highly complex protein mixtures by complementary chromatographic separation principles. Here, we describe an integrated 3D-nano-HPLC/nano-electrospray ionization quadrupole time-of-flight mass spectrometry system that allows an enzymatic digestion of proteins followed by an enrichment and subsequent separation of the created peptide mixtures. The online 3D-nano-HPLC system is composed of a monolithic trypsin reactor in the first dimension, a monolithic affinity column with immobilized monomeric avidin in the second dimension, and a reversed phase C18 HPLC-Chip in the third dimension that is coupled to a nano-ESI-Q-TOF mass spectrometer. The 3D-LC/MS setup is exemplified for the identification of biotinylated proteins from a simple protein mixture. Additionally, we describe an online 2D-nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS setup for the enrichment, separation, and identification of cross-linked, biotinylated species from chemical cross-linking of cytochrome c and a calmodulin/peptide complex using a novel trifunctional cross-linker with two amine-reactive groups and a biotin label.

Application of UPLC-QTOF-MS in MSE mode for the rapid and precise identification of alkaloids in goldenseal (Hydrastis canadensis)

Here, we describe a new application of ultra-performance liquid chromatography coupled with an electrospray ionization quadrupole time-of-flight mass spectrometry operating in MS^E mode (UPLC-QTOF-MS^E) for the sensitive, fast, and effective characterization of alkaloids in goldenseal (Hydrastis canadensis). This approach allowed identification of alkaloids using a cyclic low and high collision energy spectral acquisition mode providing simultaneous accurate precursor and fragment ion mass information. A total of 45 compounds were separated and 40 of them characterized including one new compound and 7 identified for the first time in goldenseal. The spectral data obtained using this method is comparable to those obtained by conventional LC-MSⁿ. However, the UPLC-QTOF-MS^E method offers high chromatographic resolution with structural characterization facilitated by accurate mass measurement in both MS and MS/MS modes in a single analytical run; this makes it suitable for the rapid analysis and screening of alkaloids in plant extracts.

Compressive Mass Analysis on Quadrupole Ion Trap Systems

Conventionally, quadrupole ion trap mass spectrometers eject ions of different mass-to-charge ratio (m/z) in a sequential fashion by performing a scan of the rf trapping voltage amplitude. Due to the inherent sparsity of most mass spectra, the detector measures no signal for much of the scan time. By exploiting this sparsity property, we propose a new compressive and multiplexed mass analysis approach—multi Resonant Frequency Excitation (mRFE) ejection. This new approach divides the mass spectrum into several mass subranges and detects all the subrange spectra in parallel for increased mass analysis speed. Mathematical estimation of standard mass spectrum is demonstrated while statistical classification on the parallel measurements remains viable because of the sparse nature of the mass spectra. This method can reduce mass analysis time by a factor of 3–6 and increase system duty cycle by 2×. The combination of reduced analysis time and accurate compound classification is demonstrated in a commercial quadrupole ion trap (QIT) system.

Elemental Composition Validation from Stored Waveform Inverse Fourier Transform (SWIFT) Isolation FT-ICR MS Isotopic Fine Structure

Elemental composition assignment confidence in mass spectrometry is typically assessed by monoisotopic mass accuracy. For a given mass accuracy, resolution and detection of other isotopologues can further narrow the number of possible elemental compositions. However, such measurements require ultrahigh resolving power and high dynamic range, particularly for compounds containing low numbers of nitrogen and oxygen (both ¹⁵N and ¹⁸O occur at less than 0.4 % natural abundance). Here, we demonstrate validation of molecular formula assignment from isotopic fine structure, based on ultrahigh resolution broadband Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Dynamic range is enhanced by external quadrupole and internal stored waveform inverse Fourier transform (SWIFT) isolation to facilitate detection of low abundance heavy atom isotopologues.

Factorial Experimental Designs Elucidate Significant Variables Affecting Data Acquisition on a Quadrupole Orbitrap Mass Spectrometer

Instrument parameter values for a quadrupole Orbitrap mass spectrometer were optimized for performing global proteomic analyses. Fourteen factors were evaluated for their influence on data-dependent acquisition with an emphasis on both the rate of sequencing and spectral quality by maximizing two individually tested response variables (unique peptides and protein groups). Of the 14 factors, 12 factors were assigned significant contrast values (P?<?0.05) for both response variables. Fundamentally, when optimizing parameters, a balance between spectral quality and duty cycle needs to be reached in order to maximize proteome coverage. This is especially true when using a data-dependent approach for sequencing complex proteomes. For example, maximum ion injection time, automatic gain control settings, and minimum threshold settings for triggering MS/MS isolation and activation all heavily influence ion signal, the number of spectra collected, and spectral quality. To better assess the effect these parameters have on data acquisition, all MS/MS data were parsed according to ion abundance by calculating the percent of the AGC target reached for each MS/MS event and then compared with successful peptide-spectrum matches. This proved to be an effective approach for understanding the effect of ion abundance on successful peptide-spectrum matches and establishing minimum ion abundance thresholds for triggering MS/MS isolation and activation.

The Mechanism of 2-Furaldehyde Formation from d-Xylose Dehydration in the Gas Phase. A Tandem Mass Spectrometric Study

The mechanism of reactions occurring in solution can be investigated also in the gas phase by suited mass spectrometric techniques, which allow to highlight fundamental mechanistic features independent of the influence of the medium and to clarifying controversial hypotheses proposed in solution studies. In this work, we report a gas-phase study performed by electrospray triple stage quadrupole mass spectrometry (ESI-TSQ/MS) on the dehydration of d-xylose, leading mainly to the formation of 2-furaldehyde (2-FA). It is generally known in carbohydrate chemistry that the thermal acid catalyzed dehydration of pentoses leads to the formation of 2-FA, but several aspects on the solution-phase mechanism are controversial. Here, gaseous reactant ions corresponding to protonated xylose molecules obtained from ESI of a solution containing d-xylose and ammonium acetate as protonating reagent were allowed to undergo collisionally activated decomposition (CAD) into the triple stage quadrupole analyzer. The product ion mass spectra of protonated xylose are characterized by the presence of ionic intermediates arising from xylose dehydration, which were structurally characterized by their fragmentation patterns. As expected, the xylose triple dehydration leads to the formation of the ion at m/z 97, corresponding to protonated 2-FA. On the basis of mass spectrometric evidences, we demonstrated that in the gas phase, the formation of 2-FA involves protonation at the OH group bound to the C1 atom of the sugar, the first ionic intermediate being characterized by a cyclic structure. Finally, energy resolved product ion mass spectra allowed to obtain information on the energetic features of the d-xylose→2-FA conversion.

Radical polymerization of methyl methacrylate with 2,2,3-triphenylpropanoic acid as an initiator

An unsymmetrical compound, 2,2,3-triphenylpropanoic acid (TPPA), was successfully prepared from phenyllithium, 1,1-diphenylethylene (DPE), gas carbon dioxide (CO₂), and aqueous standard solution of hydrochloric acid with LiCl deprivation. Characterization of the compound was performed by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. The polymerization of methyl methacrylate (MMA) was performed in the presence of TPPA at 95 °C. The free radicals obtained were characterized by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS). Gel permeation chromatography (GPC) traces of the average molecular weight of poly(MMA) (PMMA) showed a series of translations with increasing time. The average molecular weight of PMMA indicated narrow polydispersity, and an approximately linear relationship was found between ln ([M]₀/[M]) and polymerization time.

MS/MS Fragmentation Behavior Study of meso-Phenylporphyrinoids Containing Nonpyrrolic Heterocycles and meso-Thienyl-Substituted Porphyrins

Free base and cobalt(II) complexes of six meso-tetraphenylporphyrinoids containing nonpyrrolic heterocycles and of three meso-thienylporphyrins were investigated using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Their fragmentation was studied in a quadrupole ion trap as a function of the porphyrinoid macrocycle structure and compared with the fragmentation behavior of the benchmark compound meso-tetraphenylporphyrin. In situ oxidation of the neutral cobalt(II) complexes under ESI conditions produced singly charged cobalt(III) porphyrinoid ions; the free bases were ionized by protonation. For the porphyrinoids with an intact porphyrin core, the major fragmentation pathways observed were the losses of the meso-substituent (for meso-phenyl groups) and characteristic fragmentations of one or more meso-substituents (for the meso-thienyl group). Complex fragmentation pathways were observed for porphyrinoids with modifications to the porphyrin core but chemically reasonable structures could be assigned to most fragments, thus delineating general patterns for the behavior of pyrrole-modified porphyrins under CID conditions.

Implementation of Dipolar Resonant Excitation for Collision Induced Dissociation with Ion Mobility/Time-of-Flight MS

An ion mobility/time-of-flight mass spectrometer (IMS/TOF MS) platform that allows for resonant excitation collision induced dissociation (CID) is presented. Highly efficient, mass-resolved fragmentation without additional excitation of product ions was accomplished and over-fragmentation common in beam-type CID experiments was alleviated. A quadrupole ion guide was modified to apply a dipolar AC signal across a pair of rods for resonant excitation. The method was characterized with singly protonated methionine enkephalin and triply protonated peptide angiotensin I, yielding maximum CID efficiencies of 44 % and 84 %, respectively. The Mathieu q_x,y parameter was set at 0.707 for these experiments to maximize pseudopotential well depths and CID efficiencies. Resonant excitation CID was compared with beam-type CID for the peptide mixture. The ability to apply resonant waveforms in mobility-resolved windows is demonstrated with a peptide mixture yielding fragmentation over a range of mass-to-charge (m/z) ratios within a single IMS-MS analysis.

Boundaries of Mass Resolution in Native Mass Spectrometry

Over the last two decades, native mass spectrometry (MS) has emerged as a valuable tool to study intact proteins and noncovalent protein complexes. Studied experimental systems range from small-molecule (drug)–protein interactions, to nanomachineries such as the proteasome and ribosome, to even virus assembly. In native MS, ions attain high m/z values, requiring special mass analyzers for their detection. Depending on the particular mass analyzer used, instrumental mass resolution does often decrease at higher m/z but can still be above a couple of thousand at m/z 5000. However, the mass resolving power obtained on charge states of protein complexes in this m/z region is experimentally found to remain well below the inherent instrument resolution of the mass analyzers employed. Here, we inquire into reasons for this discrepancy and ask how native MS would benefit from higher instrumental mass resolution. To answer this question, we discuss advantages and shortcomings of mass analyzers used to study intact biomolecules and biomolecular complexes in their native state, and we review which other factors determine mass resolving power in native MS analyses. Recent examples from the literature are given to illustrate the current status and limitations.

Rapid on-site TLC–SERS detection of four antidiabetes drugs used as adulterants in botanical dietary supplements

A novel facile method has been established for rapid on-site detection of antidiabetes chemicals used to adulterate botanical dietary supplements (BDS) for diabetes. Analytes and components of pharmaceutical matrices were separated by thin-layer chromatography (TLC) then surface-enhanced Raman spectroscopy (SERS) was used for qualitative identification of trace substances on the HPTLC plate. Optimization and standardization of the experimental conditions, for example the method used for preparation of silver colloids, the mobile phase, and the concentration of colloidal silver, resulted in a very robust and highly sensitive method which enabled successful detection when the amount of adulteration was as low as 0.001 % (w/w). The method was also highly selective, enabling successful identification of some chemicals in extremely complex herbal matrices. The established TLC–SERS method was used for analysis of real BDS used to treat diabetes, and the results obtained were verified by liquid chromatography–triple quadrupole mass spectrometry (LC–MS–MS). The study showed that TLC–SERS could be used for effective separation and detection of four chemicals used to adulterate BDS, and would have good prospects for on-site qualitative screening of BDS for adulterants.

The Effective Temperature of Ions Stored in a Linear Quadrupole Ion Trap Mass Spectrometer

The extent of internal energy deposition into ions upon storage, radial ejection, and detection using a linear quadrupole ion trap mass spectrometer is investigated as a function of ion size (m/z 59 to 810) using seven ion-molecule thermometer reactions that have well characterized reaction entropies and enthalpies. The average effective temperatures of the reactants and products of the ion-molecule reactions, which were obtained from ion-molecule equilibrium measurements, range from 295 to 350 K and do not depend significantly on the number of trapped ions, m/z value, ion trap q _z value, reaction enthalpy/entropy, or the number of vibrational degrees of freedom for the seven reactions investigated. The average of the effective temperature values obtained for all seven thermometer reactions is 318?±?23 K, which indicates that linear quadrupole ion trap mass spectrometers can be used to study the structure(s) and reactivity of ions at near ambient temperature.

Combination of pentafluorophenylhydrazine derivatization and isotope dilution LC-MS/MS techniques for the quantification of apurinic/apyrimidinic sites in cellular DNA

Apurinic/apyrimidinic (AP) sites are common DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and base-excision repair mechanisms of the modified bases. Due to the strong association of AP site formation with physically/chemically induced DNA damage, quantifying AP sites provides important information for risk assessment of exposure to genotoxins and oxidative stress. However, rigorous quantification of AP sites in DNA has been hampered by technical problems relating to the sensitivity and selectivity of existing analytical methods. We have developed a new isotope dilution liquid chromatography–coupled tandem mass spectrometry (LC-MS/MS) method for the rigorous quantification of AP sites in genomic DNA. The method entails enzymatic digestion of AP site-containing DNA by endo- and exonucleases, derivatization with pentafluorophenylhydrazine (PFPH), addition of an isotopically labeled PFPH derivative as internal standard, and quantification by LC-MS/MS. The combination of PFPH derivatization with LC-MS/MS analysis on a triple quadrupole mass spectrometer allows for sensitive and selective quantification of AP sites in DNA at a detection limit of 6.5 fmol, corresponding to 4 AP sites/10⁹ nt in 5 μg of DNA, which is at least ten times more sensitive than existing analytical methods. The protocol was validated by AP site-containing oligonucleotides and applied in quantifying methyl methanesulfonate-induced formation of AP sites in cellular DNA.

Multiplex Mass Spectrometric Imaging with Polarity Switching for Concurrent Acquisition of Positive and Negative Ion Images

We have recently developed a multiplex mass spectrometry imaging (MSI) method which incorporates high mass resolution imaging and MS/MS and MS³ imaging of several compounds in a single data acquisition utilizing a hybrid linear ion trap-Orbitrap mass spectrometer (Perdian and Lee, Anal. Chem. 82, 9393–9400, 2010). Here we extend this capability to obtain positive and negative ion MS and MS/MS spectra in a single MS imaging experiment through polarity switching within spiral steps of each raster step. This methodology was demonstrated for the analysis of various lipid class compounds in a section of mouse brain. This allows for simultaneous imaging of compounds that are readily ionized in positive mode (e.g., phosphatidylcholines and sphingomyelins) and those that are readily ionized in negative mode (e.g., sulfatides, phosphatidylinositols and phosphatidylserines). MS/MS imaging was also performed for a few compounds in both positive and negative ion mode within the same experimental set-up. Insufficient stabilization time for the Orbitrap high voltage leads to slight deviations in observed masses, but these deviations are systematic and were easily corrected with a two-point calibration to background ions.

Data-Independent Microbial Metabolomics with Ambient Ionization Mass Spectrometry

Atmospheric ionization methods are ideally suited for prolonged MS/MS analysis. Data-independent MS/MS is a complementary technique for analysis of biological samples as compared to data-dependent analysis. Here, we pair data-independent MS/MS with the ambient ionization method nanospray desorption electrospray ionization (nanoDESI) for untargeted analysis of bacterial metabolites. Proof-of-principle data and analysis are illustrated by sampling Bacillus subtilis and Pseudomonas aeruginosa directly from Petri dishes. We found that this technique enables facile comparisons between strains via MS and MS/MS plots which can be translated to chemically informative molecular maps through MS/MS networking. The development of novel techniques to characterize microbial metabolites allows rapid and efficient analysis of metabolic exchange factors. This is motivated by our desire to develop novel techniques to explore the role of interspecies interactions in the environment, health, and disease. This is a contribution to honor Professor Catherine C. Fenselau in receiving the prestigious ASMS Award for a Distinguished Contribution in Mass Spectrometry for her pioneering work on microbial mass spectrometry.

Structural Characterization of Chlorophyll-a by High Resolution Tandem Mass Spectrometry

A high resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometer is used for characterizing the fragmentation of chlorophyll-a. Three tandem mass spectrometry (MS/MS) techniques, including electron-induced dissociation (EID), collisionally activated dissociation (CAD), and infrared mutiphoton dissociation (IRMPD) are applied to the singly protonated chlorophyll-a. Some previously unpublished fragments are identified unambiguously by utilizing high resolution and accurate mass value provided by the FTICR mass spectrometer. According to this research, the two long aliphatic side chains are shown to be the most labile parts, and favorable cleavage sites are proposed. Even though similar fragmentation patterns are generated by all three methods, there are much more abundant peaks in EID and IRMPD spectra. The similarities and differences are discussed in detail. Comparatively, cleavage leading to odd electron species and H^? loss both seem more common in EID experiments. Extensive loss of small side groups (e.g., methyl and ethyl) next to the macrocyclic ring was observed. Coupling the high performance FTICR mass spectrometer with contemporary MS/MS techniques, especially IRMPD and EID, proved to be very promising for the structural characterization of chlorophyll, which is also suitable for the rapid and accurate structural investigation of other singly charged porphyrinic compounds.      

Study of Ozone-Initiated Limonene Reaction Products by Low Temperature Plasma Ionization Mass Spectrometry

Limonene and its ozone-initiated reaction products were investigated in situ by low temperature plasma (LTP) ionization quadrupole time-of-flight (QTOF) mass spectrometry. Helium was used as discharge gas and the protruding plasma generated ~850 ppb ozone in front of the glass tube by reaction with the ambient oxygen. Limonene applied to filter paper was placed in front of the LTP afterglow and the MS inlet. Instantly, a wide range of reaction products appeared, ranging from m/z 139 to ca. 1000 in the positive mode and m/z 115 to ca. 600 in the negative mode. Key monomeric oxidation products including levulinic acid, 4-acetyl-1-methylcyclohexene, limonene oxide, 3-isopropenyl-6-oxo-heptanal, and the secondary ozonide of limonene could be identified by collision-induced dissociation. Oligomeric products ranged from the nonoxidized dimer of limonene (C₂₀H₃₀) and up to the hexamer with 10 oxygen atoms (C₆₀H₉₀O₁₀). The use of LTP for in situ ozonolysis and ionization represents a new and versatile approach for the assessment of ozone-initiated terpene chemistry.

Protected Amine Labels: A Versatile Molecular Scaffold for Multiplexed Nominal Mass and Sub-Da Isotopologue Quantitative Proteomic Reagents

We assemble a versatile molecular scaffold from simple building blocks to create binary and multiplexed stable isotope reagents for quantitative mass spectrometry. Termed Protected Amine Labels (PAL), these reagents offer multiple analytical figures of merit including, (1) robust targeting of peptide N-termini and lysyl side chains, (2) optimal mass spectrometry ionization efficiency through regeneration of primary amines on labeled peptides, (3) an amino acid-based mass tag that incorporates heavy isotopes of carbon, nitrogen, and oxygen to ensure matched physicochemical and MS/MS fragmentation behavior among labeled peptides, and (4) a molecularly efficient architecture, in which the majority of hetero-atom centers can be used to synthesize a variety of nominal mass and sub-Da isotopologue stable isotope reagents. We demonstrate the performance of these reagents in well-established strategies whereby up to four channels of peptide isotopomers, each separated by 4 Da, are quantified in MS-level scans with accuracies comparable to current commercial reagents. In addition, we utilize the PAL scaffold to create isotopologue reagents in which labeled peptide analogs differ in mass based on the binding energy in carbon and nitrogen nuclei, thereby allowing quantification based on MS or MS/MS spectra. We demonstrate accurate quantification for reagents that support 6-plex labeling and propose extension of this scheme to 9-channels based on a similar PAL scaffold. Finally, we provide exemplar data that extend the application of isotopologe-based quantification reagents to medium resolution, quadrupole time-of-flight mass spectrometers.

A validated assay to quantitate serotonin in lamb plasma using ultrahigh-performance liquid chromatography-tandem mass spectrometry: applications with LC/MS3

An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS) method was developed and validated for the quantification of serotonin (5-HT) in lamb plasma using [²d₄]-serotonin ([²d₄]-5-HT) as an internal standard. Charcoal-stripped human plasma was used as the blank matrix during validation, and 5-HT was quantitated using selected reaction monitoring. The UHPLC/MS/MS system consisted of an Agilent 1290 Infinity ultrahigh-performance liquid chromatograph coupled with an AB SCIEX QTRAP® 5500 hybrid linear ion trap triple quadrupole mass spectrometer. The method was validated for accuracy, precision, linearity, lower limit of quantification (LLOQ), selectivity, and other parameters. The LLOQ was 1.0 ng/mL, requiring 100 μL of sample. The method was applied to monitor the 5-HT levels in lamb plasma after the administration of fluoxetine. Tandem mass spectrometry cubed (MS³) experiments were also performed to investigate the fragmentation pattern of 5-HT and [²d₄]-5-HT. A liquid chromatography-MS³ (LC/MS³) method was developed, and the UHPLC/MS/MS and the LC/MS³ methods were compared for performance.

Estimation of Activation Energy from the Survival Yields: Fragmentation Study of Leucine Enkephalin and Polyethers by Tandem Mass Spectrometry

A simple collision model for multiple collisions occurring in quadrupole type mass spectrometers was derived and tested with leucine enkaphalin a common mass spectrometric standard with well-characterized properties. Implementation of the collision model and Rice-Ramsperger-Kassel-Marcus (RRKM) algorithm into a spreadsheet software allowed a good fitting of the calculated data to the experimental survival yield (SY) versus collision energy curve. In addition, fitting also ensured to estimate the efficiencies of the kinetic to internal energy conversion for Leucine enkephalin in quadrupole-time-of-flight and triple quadrupole instruments. It was observed that the experimental SY versus collision energy curves for the leucine enkephalin can be described by the Rice-Ramsperger-Kassel (RRK) formalism by reducing the total degrees of freedom (DOF) to about one-fifth. Furthermore, this collision model with the RRK formalism was used to estimate the critical energy (E _o ) of lithiated polyethers, including polyethylene glycol (PEG), polypropylene glycol (PPG), and polytetrahydrofurane (PTHF) with degrees of freedom similar to that of leucine enkephalin. Applying polyethers with similar DOF provided the elimination of the effect of DOF on the unimolecular reaction rate constant. The estimated value of E _o for PEG showed a relatively good agreement with the value calculated by high-level quantum chemical calculations reported in the literature. Interestingly, it was also found that the E _o values for the studied polyethers were similar.