Rapid Denaturing Organic Digestion Method for Targeted Protein Identification and Characterization.

Bottom-up mass spectrometry-based protein analysis methods employing protease digestion are routinely used to identify and characterize proteins with high specificity and sensitivity. Method performance is generally measured by sequence coverage capability and the total number of characteristic peptides identified, when compared to predicted databases. Limitations to commonly used solvent-based digestion methods currently employed include long digestion times (18-24 h or more), leading to protease autolysis, which also precludes automation, decreases sensitivity, and increases both intra- and inter-day performance variability. This report describes the development and validation of a simple, 5 min tryptic denaturing organic digestion (DOD) method for use with tandem mass spectrometry in bottom-up protein identification and characterization. It has been evaluated across select protein toxins and diagnostic clinical protein targets, substantially improving digestion performance when compared to other solution-based and enzyme-immobilized methods. The method was compared to two currently used bottom-up methods, the 24 h filter-aided sample prep (FASP) and Flash Digest (1 and 4 h) methods. Single proteins used to compare the methods included the ricin light chain, ricin heavy chain, ricin holotoxin, serotype A Clostridium botulinum toxin, Staphylococcus enterotoxin B, ribonuclease A, and thyroglobulin. In tests, across the proteins investigated, the 5 min DOD digestion method resulted in sequence coverages ranging from 55 to 100%, with relatively high reproducibility and precision; results were better than or equal to FASP method results and were greatly enhanced when compared to Flash method results. Importantly, DOD method intra- and inter-day precision was much improved as compared to results for both FASP and Flash digestions. These data indicated that the DOD method, when compared to the FASP and Flash Digest methods, dramatically reduced digestion time, while maintaining or improving the ability to detect and characterize targeted proteins, and reduced analytical variability for tryptic digestion, resulting in markedly faster and more precise analyses.

Improved N- and C-Terminal Sequencing of Proteins by Combining Positive and Negative Ion MALDI In-Source Decay Mass Spectrometry.

The development of various ionization and fragmentation techniques has been of key importance for establishing mass spectrometry (MS) as a powerful tool for protein characterization. One example of this is matrix-assisted laser desorption/ionization (MALDI) combined with in-source decay (ISD) fragmentation that allows mapping of N- and C-terminal regions of large proteins without the need for proteolysis. Positive ion mode ISD fragments are commonly assigned in the mass region above m/z 1000, while MALDI matrix ions generally hamper the detection of smaller singly charged fragments. The ultrahigh resolving power provided by Fourier transform ion cyclotron resonance (FT-ICR) MS partially overcomes this limitation, but to further increase the detection of smaller fragments we have revisited the application of negative ion mode MALDI-ISD and found good coverage of the peptide chain termini starting from c'2 and z'2 fragment ions. For the first time, we demonstrate that the combination of negative and positive ion MALDI FT-ICR MS is a useful tool to improve the characterization of mAbs. The different specificities of the two ion modes allowed us to selectively cover the sequence of the light and heavy chains of mAbs at increased sensitivity. A comprehensive evaluation of positive and negative ion mode MALDI-ISD FT-ICR MS in the m/z range 46-13 500 showed an increased sequence coverage for three standard proteins, namely, myoglobin, SiLuLite mAb, and NIST mAb. The data obtained in the two ion modes were, in part, complementary.

Evaluation of Sibling and Twin Fragment Ions Improves the Structural Characterization of Proteins by Top-Down MALDI In-Source Decay Mass Spectrometry.

Comprehensive determination of primary sequence and identification of post-translational modifications (PTMs) are key elements in protein structural analysis. Various mass spectrometry (MS) based fragmentation techniques are powerful approaches for mapping both the amino acid sequence and PTMs; one of these techniques is matrix-assisted laser desorption/ionization (MALDI), combined with in-source decay (ISD) fragmentation and Fourier-transform ion cyclotron resonance (FT-ICR) MS. MALDI-ISD MS protein analysis involves only minimal sample preparation and does not require spectral deconvolution. The resulting MALDI-ISD MS data is complementary to electrospray ionization-based MS/MS sequencing readouts, providing knowledge on the types of fragment ions is available. In this study, we evaluate the isotopic distributions of z' ions in protein top-down MALDI-ISD FT-ICR mass spectra and show why these distributions can deviate from theoretical profiles as a result of co-occurring and isomeric z and y-NH3 ions. Two synthetic peptides, containing either normal or deuterated alanine residues, were used to confirm the presence and unravel the identity of isomeric z and y-NH3 fragment ions ("twins"). Furthermore, two reducing MALDI matrices, namely 1,5-diaminonaphthalene and N-phenyl-p-phenylenediamine were applied that yield ISD mass spectra with different fragment ion distributions. This study demonstrates that the relative abundance of isomeric z and y-NH3 ions requires consideration for accurate and confident assignments of z' ions in MALDI-ISD FT-ICR mass spectra.

Structural Analysis of Monoclonal Antibodies by Ultrahigh Resolution MALDI In-Source Decay FT-ICR Mass Spectrometry.

The emergence of complex protein therapeutics in general and monoclonal antibodies (mAbs) in particular have stimulated analytical chemists to develop new methods and strategies for their structural characterization. Mass spectrometry plays a key role in providing information on the primary amino acid sequence, post-translational modifications, and other structure characteristics that must be monitored during the manufacturing process and subsequent quality control assessment. In this study, we present a novel method that allows structural characterization of mAbs based on MALDI in-source decay (ISD) fragmentation, coupled with Fourier transform ion cyclotron resonance (FT-ICR) MS. The method benefits from higher resolution of absorption mode FT mass spectra, compared to magnitude mode, which enables simultaneous identification of ISD fragments from both the heavy and light chains with a higher confidence in a wide mass range up to m/ z 13 500. This method was applied to two standard mAbs, namely NIST mAb and trastuzumab, in preparation for method application in an interlaboratory study on mAbs structural analysis coordinated by the Consortium for Top-Down Proteomics. Extensive sequence coverage was obtained from the middle-down analysis (IdeS- and GingisKHAN-digested mAbs) that complemented the top-down analysis of intact mAbs. In addition, MALDI FT-ICR MS of IdeS-digested mAbs allowed isotopic-level profiling of proteoforms with regard to heavy chain N-glycosylation.

A Light-Activated Acyl Carrier Protein "Trap" for Intermediate Capture in Type†II Iterative Polyketide Biocatalysis.

A discrete acyl carrier protein (ACP) bearing a photolabile nonhydrolysable carba(dethia) malonyl pantetheine cofactor was chemoenzymatically prepared and utilised for the trapping of biosynthetic polyketide intermediates following light activation. From the in vitro assembly of the polyketides SEK4 and SEK4b, by the type II actinorhodin "minimal" polyketide synthase (PKS), a range of putative ACP-bound diketides, tetraketides, pentaketides and hexaketides were identified and characterised by FT-ICR-MS, providing direct insights on active site accessibility and substrate processing for this enzyme class.

Native-MS Analysis of Monoclonal Antibody Conjugates by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Antibody-drug conjugates (ADCs) are an important class of therapeutic molecule currently being used to treat HER2-positive metastatic breast cancer, relapsed or refractory Hodgkin lymphoma, systemic anaplastic large cell lymphoma, relapsed or refractory B-cell precursor acute lymphoblastic leukemia, and acute myeloid leukemia. An ADC typically consists of a small molecule or peptide-based cytotoxic moiety covalently linked, via lysine or cysteine residues, to a monoclonal antibody (mAb) scaffold. Mass spectrometric (MS) characterization of these molecules affords highly accurate molecular weight (MW) and drug-to-antibody ratio (DAR) determination and is typically performed using orthogonal acceleration time-of-flight (oa-ToF) analyzers and more recently, Orbitrap instruments. Herein we describe for the first time the use of a 15 T solariX Fourier transform ion cyclotron mass spectrometer to characterize an IgG1 mAb molecule conjugated with biotin via native lysine and cysteine residues, under native-MS and solution conditions. The cysteine-biotin conjugates remained fully intact, demonstrating the ability of the FT-ICR to maintain the noncovalent interactions and efficiently transmit labile protein complexes. Native-MS was acquired and is displayed in magnitude mode using a symmetric Hann apodization function. Baseline separation is achieved on all covalent biotin additions, for each charge state, for both the lysine- and cysteine-biotin conjugates. Average DAR values obtained by native-MS for the lysine conjugate are compared to those derived by denaturing reversed phase liquid chromatography using an oa-ToF MS system (1.56 ± 0.02 versus 2.24 ± 0.02 for the 5 equivalent and 3.99 ± 0.09 versus 4.43 ± 0.01 for the 10 equivalent, respectively). Increased DAR value accuracy can be obtained for the higher biotin-load when using standard ESI conditions as opposed to nanoESI native-MS conditions.

An ambient detection system for visualization of charged particles generated with ionization methods at atmospheric pressure.

RATIONALE: With the current state-of-the-art detection of ions only taking place under vacuum conditions, active pixel detectors that operate under ambient conditions are of particular interest. These detectors are ideally suited to study and characterize the charge distributions generated by ambient ionization sources. METHODS: The direct imaging capabilities of the active pixel detector are used to investigate the spatial distributions of charged droplets generated by three ionization sources, named electrospray ionization (ESI), paper spray ionization (PSI) and surface acoustic wave nebulization (SAWN). The ionization spray (ESI/PSI) and ionization plume (SAWN) originating from each source are directly imaged. The effect of source parameters such as spray voltage for ESI and PSI, and the angle of the paper spray tip on the charge distributions, is investigated. Two types of SAWN liquid interface, progressive wave (PW) and standing wave (SW), are studied. RESULTS: Direct charge detection under ambient conditions is demonstrated using an active pixel detector. Direct charge distributions are obtained of weak, homogeneous/focused and dispersed spray plumes by applying low, intermediate and high spray potentials, respectively, for ESI. Spray plume footprints obtained for various angles of PSI shows the possibility to focus the ion beam as a function of the paper angle. Differences between two designs of the SAWN interface are determined. Droplet charge flux changes are illustrated in a way similar to a total ion chromatogram. CONCLUSIONS: The use of this active pixel detector allows the rapid characterization and optimization of different ambient ionization sources without the actual use of a mass spectrometer. Valuable illustrations are obtained of changes in spatial distribution and number of charges detected for ESI, PSI and SAWN ion plumes. Copyright © 2015 John Wiley & Sons, Ltd.

Producing Isotopic Distribution Models for Fully Apodized Absorption Mode FT-MS.

Isotopic distributions are frequently used as part of the peak assignment process in the processing of mass spectra. The best methods for producing accurate peak assignments must account for the peak shape and resolving power. In other words, the full profile of the isotopic distribution is important. Conventional methods for modeling isotopic distributions generally assume a peak profile that is not applicable to fully apodized absorption mode spectra because the peak shapes in these spectra are distinctly different from those seen in normal (i.e., magnitude mode) spectra. We present results illustrating this problem and describe a method for producing more accurate isotopic distribution models for this class of spectra.

Absorption mode Fourier transform mass spectrometry with no baseline correction using a novel asymmetric apodization function.

RATIONALE: Absorption mode Fourier transform ion cyclotron resonance (FTICR) mass spectra offer significant benefits in terms of spectral resolution, signal-to-noise (S/N) ratio and measured mass accuracy. However, to date, methods for producing absorption mode spectra have created an undesirable baseline deviation as a consequence of FFT artifacts, resulting in interference of the frequency side-lobes of intense peaks. Methods for fitting and removing this deviation have been developed, but these are computationally intensive, slow and can be unreliable in practice. METHODS: We have developed an approach for producing FTICR mass spectra which uses a new apodization approach to produce spectra which do not exhibit baseline deviation, whilst maintaining all the normal absorption mode benefits. This method involves the use of 'full' apodization function, replacing the more common Hann or half Hann functions, and where the user can control the position of the function maximum expressed as a fraction (F) of the transient length. RESULTS: Absorption mode spectra produced using the new apodization function we propose provide all the normal benefits but do not exhibit baseline deviation that must be corrected prior to spectral interpretation. Additionally, varying the value of the F parameter allows users additional control over the compromise between the spectral resolving power and the S/N ratio. This is particularly beneficial in spectra with pronounced amplitude changes during the recording of the transient (detection). CONCLUSIONS: The use of a 'full' apodization function, which may be asymmetric, prior to zero-padding and Fourier transformation, allows the production of absorption mode spectra which do not suffer from baseline deviation. Hence, it is no longer necessary to apply a baseline deviation correction in post processing, providing a significant performance advantage.

Producing absorption mode Fourier transform ion cyclotron resonance mass spectra with non-quadratic phase correction functions.

RATIONALE: Previously described methods for producing absorption mode Fourier transform ion cyclotron resonance (FTICR) mass spectra have all relied on the phase correction function being quadratic. This assumption has been found to be invalid for some instruments and spectra and so it has not been possible to produce absorption mode spectra for these cases. METHODS: The Autophaser algorithm has been adapted to allow nth order polynomial phase correction functions to be optimized. The data was collected on a modified Thermo LTQ FTICR mass spectrometer, using electrospray ionization and a novel ICR cell design (NADEL). Peak assignment and mass calibration were undertaken using the pyFTMS framework. RESULTS: An nth-order phase correction function has been used to produce an absorption mode mass spectrum of the maltene fraction of a crude oil sample which was not possible using the previous assumption that the phase correction function must be quadratic. Data processing for this spectrum in absorption mode has shown the expected benefits in terms of increasing the number of assigned peaks and also improving the mass accuracy (i.e. confidence) of the assignments. CONCLUSIONS: It is possible to phase-correct time-domain data in FTICRMS to yield absorption mode mass spectra representation even when the data does not correspond to the theoretical quadratic phase correction function predicted by previous studies. This will allow a larger proportion of spectra to be processed in absorption mode.

Autophaser: an algorithm for automated generation of absorption mode spectra for FT-ICR MS.

Phase correction of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry data allows the spectra to be presented in absorption mode. Absorption mode spectra offer superior mass resolving power (up to a factor of 2), mass accuracy, and sensitivity over the conventional magnitude mode. Hitherto, the use of absorption mode in FT-ICR mass spectrometry has required either specially adapted instrumentation or a manually intensive process of phase correction or has ignored the potentially significant effects of image charge and the associated frequency shifts. Here we present an algorithm that allows spectra recorded on unadapted FT-ICR mass spectrometers to be phase corrected, their baseline deviations removed, and then an absorption mode spectrum presented in an automated manner that requires little user interaction.

Absorption mode FTICR mass spectrometry imaging.

Fourier transform ion cyclotron resonance mass spectrometry offers the highest mass resolving power for molecular imaging experiments. This high mass resolving power ensures that closely spaced peaks at the same nominal mass are resolved for proper image generation. Typically higher magnetic fields are used to increase mass resolving power. However, a gain in mass resolving power can also be realized by phase correction of the data for absorption mode display. In addition to mass resolving power, absorption mode offers higher mass accuracy and signal-to-noise ratio over the conventional magnitude mode. Here, we present the first use of absorption mode for Fourier transform ion cyclotron resonance mass spectrometry imaging. The Autophaser algorithm is used to phase correct each spectrum (pixel) in the image, and then, these parameters are used by the Chameleon work-flow based data processing software to generate absorption mode "Datacubes" for image and spectral viewing. Absorption mode reveals new mass and spatial features that are not resolved in magnitude mode and results in improved selected ion image contrast.

Improved optimization of the Fourier transform ion cyclotron resonance mass spectrometry phase correction function using a genetic algorithm.

RATIONALE: Fourier Transform Ion Cyclotron Resonance mass spectra exhibit improved resolving power, mass accuracy and signal-to-noise ratio when presented in absorption mode; a process which requires calculation of a phase correction function. Mass spectrometric images can contain many thousands of pixels; hence methods of decreasing the time required to solve for a phase correction function will result in significant improvements in this application. METHODS: A genetic algorithm approach for optimizing the phase correction function has been developed and compared with a previously described convergent iteration technique. RESULTS: The genetic algorithm method has been shown to offer a five-fold improvement in processing speed compared with the previous iterative approach used in the Autophaser algorithm, while maintaining the levels of accuracy. This translates to an 11 hour improvement in processing for a 20 000 pixel mass spectrometric image. CONCLUSIONS: The genetic algorithm method described in this manuscript offers significant processing speed advantages over the previously described convergent iteration technique. This improvement is key to allowing the future routine use of absorption mode mass spectrometric images.

Determining the Location of the α-Synuclein Dimer Interface Using Native Top-Down Fragmentation and Isotope Depletion-Mass Spectrometry.

α-Synuclein (αSyn), a 140-residue intrinsically disordered protein, comprises the primary proteinaceous component of pathology-associated Lewy body inclusions in Parkinson's disease (PD). Due to its association with PD, αSyn is studied extensively; however, the endogenous structure and physiological roles of this protein are yet to be fully understood. Here, ion mobility-mass spectrometry and native top-down electron capture dissociation fragmentation have been used to elucidate the structural properties associated with a stable, naturally occurring dimeric species of αSyn. This stable dimer appears in both wild-type (WT) αSyn and the PD-associated variant A53E. Furthermore, we integrated a novel method for generating isotopically depleted protein into our native top-down workflow. Isotope depletion increases signal-to-noise ratio and reduces the spectral complexity of fragmentation data, enabling the monoisotopic peak of low abundant fragment ions to be observed. This enables the accurate and confident assignment of fragments unique to the αSyn dimer to be assigned and structural information about this species to be inferred. Using this approach, we were able to identify fragments unique to the dimer, which demonstrates a C-terminal to C-terminal interaction between the monomer subunits. The approach in this study holds promise for further investigation into the structural properties of endogenous multimeric species of αSyn.

The present and potential future of aqueous mercury preservation: a review.

Mercury is considered to be one of the most toxic elements to humans. Due to pollution from industry and artisanal gold mining, mercury species are present globally in waters used for agriculture, aquaculture, and drinking water. This review summarises methods reported for preserving mercury species in water samples and highlights the associated hazards and issues with each. This includes the handling of acids in an uncontrolled environment, breakage of sample containers, and the collection and transport of sample volumes in excess of 1 L, all of which pose difficulties for both in situ collection and transportation. Literature related to aqueous mercury preservation from 2000-2021 was reviewed, as well as any commonly cited and relevant references. Amongst others, solid-phase extraction techniques were explored for preservation and preconcentration of total and speciated mercury in water samples. Additionally, the potential as a safe, in situ preservation and storage method for mercury species were summarised. The review highlighted that the stability of mercury is increased when adsorbed on a solid-phase and therefore the metal and its species can be preserved without the need for hazardous reagents or materials in the field. The mercury species can then be eluted upon return to a laboratory, where sensitive analytical detection and speciation methods can be better applied. Developments in solid phase extraction as a preservation method for unstable metals such as mercury will improve the quality of representative environmental data, and further improve toxicology and environmental monitoring studies.

Appropriate degree of trust: deriving confidence metrics for automatic peak assignment in high-resolution mass spectrometry.

Techniques for deriving confidence metrics for the reliability of automatically assigned elemental formulas in complex spectra, from high-resolution mass spectrometers, are described. These metrics can help an analyst to place an appropriate degree of trust in the results obtained from automated spectral analysis of, for example, natural organic materials. To provide these metrics of confidence, common mass spectrometric tests for reliability of peak assignment (mass accuracy/error, relative ion abundance, and rings-plus-double-bonds equivalence) are combined with novel confidence metrics based on the interconnectivity and consistency of a mass difference or mass defect based peak inference network and on the confidence of the initial library matches. These are shown to provide improved peak assignment confidence over manual or simple automatic assignment methods.

Use of an artificial immune system derived method for the charge state assignment of small-molecule mass spectra.

Knowing the charge state of an ion in a mass spectrum is crucial to being able to assign a formula to it. For many small-molecule peaks in complex mass spectra, the intensities of the isotopic peaks are too low to allow the charge state to be calculated from isotopic spacings, which is the basis of the conventional method of determining the charge state of an ion. A novel artificial intelligence derived method for identifying the charge state of ions, in the absence of any isotopic information or a series of charge states, has been developed using an artificial immune system approach. This technique has been tested against synthetic and real data sets and has proven successful in identifying the majority of multiply charged ions, thereby significantly improving the peak assignment rate and confidence.

Isotope Depletion Mass Spectrometry (ID-MS) for Accurate Mass Determination and Improved Top-Down Sequence Coverage of Intact Proteins.

Top-down mass spectrometry (MS) is an increasingly important technique for protein characterization. However, in many biological MS experiments, the practicality of applying top-down methodologies is still limited at higher molecular mass. In large part, this is due to the detrimental effect resulting from the partitioning of the mass spectral signal into an increasing number of isotopic peaks as molecular mass increases. Reducing the isotopologue distribution of proteins via depletion of heavy stable isotopes was first reported over 20 years ago (Marshall, A. G.; Senko, M. W.; Li, W.; Li, M.; Dillon, S., Guan, S.; Logan, T. M.. Protein Molecular Mass to 1 Da by 13C, 15N Double-Depletion and FT-ICR Mass Spectrometry. J. Am. Chem. Soc. 1997, 119, 433-434.) and has been demonstrated for several small proteins. Here we extend this approach, introducing a new highly efficient method for the production of recombinant proteins depleted in 13C and 15N and demonstrating its advantages for top-down analysis of larger proteins (up to ∼50 kDa). FT-ICR MS of isotopically depleted proteins reveals dramatically reduced isotope distributions with monoisotopic signal observed up to 50 kDa. In top-down fragmentation experiments, the reduced spectral complexity alleviates fragment-ion signal overlap, the presence of monoisotopic signals allows assignment with higher mass accuracy, and the dramatic increase in signal-to-noise ratio (up to 7-fold) permits vastly reduced acquisition times. These compounding benefits allow the assignment of ∼3-fold more fragment ions than comparable analyses of proteins with natural isotopic abundances. Finally, we demonstrate greatly increased sequence coverage in time-limited top-down experiments-highlighting advantages for top-down LC-MS/MS workflows and top-down proteomics.

Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry.

Bispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The "bi-specificity" of these complex biopharmaceuticals is a key characteristic for the development of novel and more effective therapeutic strategies. The high structural complexity of BsAbs poses a challenge to the analytical methods needed for their characterization. Modifications of the BsAb structure, resulting from enzymatic and non-enzymatic processes, further complicate the analysis. An important example of the latter type of modification is glycation, which can occur in the manufacturing process, during storage in the formulation or in vivo after application of the drug. Glycation affects the structure, function, and stability of monoclonal antibodies, and consequently, a detailed analysis of glycation levels is required. Mass spectrometry (MS) plays a key role in the structural characterization of monoclonal antibodies and top-down, middle-up and middle-down MS approaches are increasingly used for the analysis of modifications. Here, we apply a novel middle-up strategy, based on IdeS digestion and matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS, to analyze all six different BsAb subunits in a single high-resolution mass spectrum, namely two light chains, two half fragment crystallizable regions and two Fd' regions, thus avoiding upfront chromatography. This method was used to monitor glycation changes during a 168 h forced-glycation experiment. In addition, hot spot glycation sites were localized using top-down and middle-down MALDI-in-source decay FT-ICR MS, which provided complementary information compared to standard bottom-up MS.