The analysis of many hydrogen exchange (HX) experiments depends on knowledge of exchange rates expected for the unstructured protein under the same conditions. We present here some minor adjustments to previously calibrated values and a stringent test of their accuracy.
Differential or field asymmetric waveform ion mobility spectrometry (FAIMS) operating at high electric fields fully resolves isotopic isomers for a peptide with labeled residues. The naturally present isotopes, alone and together with targeted labels, also cause spectral shifts that approximately add for multiple heavy atoms. Separation qualitatively depends on the gas composition. These findings may enable novel strategies in proteomic and metabolomic analyses using stable isotope labeling.
Peptides with deamidated asparagine residues and oxidized methionine residues are often not resolved sufficiently to allow quantitation of their native and modified forms using reversed phase (RP) chromatography. The accurate quantitation of these modifications is vital in protein biotherapeutic analysis because they can affect a protein’s function, activity, and stability. We demonstrate here that hydrophilic interaction liquid chromatography (HILIC) adequately and predictably separates peptides with these modifications from their native counterparts. Furthermore, coefficients describing the extent of the hydrophilicity of these modifications have been derived and were incorporated into a previously made peptide retention prediction model that is capable of predicting the retention times of peptides with and without these modifications.
Previous reports have shown that reactions occurring in the microdroplets formed during electrospray ionization can, under the right conditions, exhibit significantly greater rates than the corresponding bulk solution-phase reactions. The observed acceleration under electrospray ionization could result from a solution-phase, a gas-phase, or an interfacial reaction. This study shows that a gas-phase ion/molecule (or ion/ion) reaction is not responsible for the observed rate enhancement in the particular case of the Fischer indole synthesis. The results show that the accelerated reaction proceeds in the microdroplets, and evidence is provided that an interfacial process is involved.
The flowing atmospheric-pressure afterglow (FAPA) source was used for the mass-spectrometric analysis of vapor samples introduced between the source and mass spectrometer inlet. Through interrupted operation of the plasma-supporting helium flow, helium consumption is greatly reduced and dynamic gas behavior occurs that was characterized by schlieren imaging. Moreover, mass spectra acquired immediately after the onset of helium flow exhibit a signal spike before declining and ultimately reaching a steady level. This initial signal appears to be due to greater interaction of sample vapor with the afterglow of the source when helium flow resumes. In part, the initial spike in signal can be attributed to a pooling of analyte vapor in the absence of helium flow from the source. Time-resolved schlieren imaging of the helium flow during on and off cycles provided insight into gas-flow patterns between the FAPA source and the MS inlet that were correlated with mass-spectral data.
A cationic degradation product, formed in solution from retinal Schiff base (RSB), is examined in the gas phase using ion mobility spectrometry, photoisomerization action spectroscopy, and collision induced dissociation (CID). The degradation product is found to be N-n-butyl-2-(β-ionylidene)-4-methylpyridinium (BIP) produced through 6π electrocyclization of RSB followed by protonation and loss of dihydrogen. Ion mobility measurements show that BIP exists as trans and cis isomers that can be interconverted through buffer gas collisions and by exposure to light, with a maximum response at λ = 420 nm.
Fungal secondary metabolites represent a rich and largely untapped source for bioactive molecules, including peptides with substantial structural diversity and pharmacological potential. As methods proceed to take a deep dive into fungal genomes, complimentary methods to identify bioactive components are required to keep pace with the expanding fungal repertoire. We developed PepSAVI-MS to expedite the search for natural product bioactive peptides and herein demonstrate proof-of-principle applicability of the pipeline for the discovery of bioactive peptides from fungal secretomes via identification of the antifungal killer toxin KP4 from Ustilago maydis P4. This work opens the door to investigating microbial secretomes with a new lens, and could have broad applications across human health, agriculture, and food safety.
Short-term glucose starvation prior to chemotherapy has the potential to preferentially weaken cancer cells, making them more likely to succumb to treatment, while protecting normal cells. In this study, we used 3D cell cultures of colorectal cancer and assessed the effects of short-term glucose starvation and chemotherapy compared to treatment of either individually. We evaluated both phenotypic changes and protein expression levels. Our findings indicate that the combined treatment results in more significant phenotypic responses, including decreased cell viability and clonogenicity. These phenotypic responses can be explained by the decreased expression of LDHA and 14-3-3 family proteins, which were found only in the combined treatment groups. This study indicates that short-term glucose starvation has the potential to increase the efficacy of current cancer treatment regimes.
The development of tandem ion mobility spectroscopy (IMS) known as IMS-IMS has led to extensive research into isomerizations of isolated molecules. Many recent works have focused on the retinal chromophore which is the optical switch used in animal vision. Here, we study a shortened derivative of the chromophore, which exhibits a rich IM spectrum allowing for a detailed analysis of its isomerization pathways, and show that the longer the chromophore is, the lower the barrier energies for isomerization are.
Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) allows data-independent fragmentation of all ions in a sample and correlation of fragment ions to their precursors through the modulation of precursor ion cyclotron radii prior to fragmentation. Previous results show that implementation of 2D FT-ICR MS with infrared multi-photon dissociation (IRMPD) and electron capture dissociation (ECD) has turned this method into a useful analytical tool. In this work, IRMPD tandem mass spectrometry of calmodulin (CaM) has been performed both in one-dimensional and two-dimensional FT-ICR MS using a top-down and bottom-up approach. 2D IRMPD FT-ICR MS is used to achieve extensive inter-residue bond cleavage and assignment for CaM, using its unique features for fragment identification in a less time- and sample-consuming experiment than doing the same thing using sequential MS/MS experiments.
Vitamin D compounds are secosteroids, which are best known for their role in bone health. More recent studies have shown that vitamin D metabolites and catabolites such as dihydroxylated species (e.g., 1,25- and 24,25-dihydroxyvitamin D3) play key roles in the pathologies of various diseases. Identification of these isomers by mass spectrometry is challenging and currently relies on liquid chromatography, as the isomers exhibit virtually identical product ion spectra under collision induced dissociation conditions. Here, we developed a simple MALDI-CID method that utilizes ion activation of reactive analyte/matrix adducts to distinguish isomeric dihydroxyvitamin D3 species, without the need for chromatography separation or chemical derivatization techniques. Specifically, reactive 1,5-diaminonaphthalene adducts of dihydroxyvitamin D3 compounds formed during MADI were activated and specific cleavages in the secosteroid’s backbone structure were achieved that produced isomer-diagnostic fragment ions.
A high performance liquid chromatograph (HPLC)was interfaced to an atmospheric drift tube ion mobility time of flight mass spectrometry. The power of multidimensional separation was demonstrated using chili pepper extracts. The ambient pressure drift tube ion mobility provided high resolving powers up to 166 for the HPLC eluent. With implementation of Hadamard transform (HT), the duty cycle for the ion mobility drift tube was increased from less than 1% to 50%, and the ion transmission efficiency was improved by over 200 times compared with pulsed mode, improving signal to noise ratio 10 times. HT ion mobility and TOF mass spectrometry provide an additional dimension of separation for complex samples without increasing the analysis time compared with conventional HPLC.
A modified Kendrick Mass Defect (KMD) analysis was applied to the analysis of polycyclic aromatic hydrocarbons (PAHs) and fullerenes in the diffusion flame from a handheld butane torch.
We present a simple algorithm for robust and unsupervised peak detection by determining a noise threshold in isotopically resolved mass spectrometry data. Solving this problem will greatly reduce the subjective and time-consuming manual picking of mass spectral peaks and so will prove beneficial in many research applications. The Autopiquer approach uses autocorrelation to test for the presence of (isotopic) structure in overlapping windows across the spectrum. Within each window, a noise threshold is optimized to remove the most unstructured data, whilst keeping as much of the (isotopic) structure as possible. This algorithm has been successfully demonstrated for both peak detection and spectral compression on data from many different classes of mass spectrometer and for different sample types, and this approach should also be extendible to other types of data that contain regularly spaced discrete peaks.
We provide modeling and experimental data describing the dominant ion-loss mechanisms for differential mobility spectrometry (DMS). Ion motion is considered from the inlet region of the mobility analyzer to the DMS exit, and losses resulting from diffusion to electrode surfaces, insufficient effective gap, ion fragmentation, and fringing field effects are considered for a commercial DMS system with 1-mm gap height. It is shown that losses due to diffusion and radial oscillations can be minimized with careful consideration of residence time, electrode spacing, gas flow rate, and waveform frequency. Fragmentation effects can be minimized by limitation of the separation field. When these parameters were optimized, fringing field effects at the DMS inlet contributed the most to signal reduction. We also describe a new DMS cell configuration that improves the gas dynamics at the mobility cell inlet. The new cell provides a gas jet that decreases the residence time for ions within the fringing field region, resulting in at least twofold increase in ion signal as determined by experimental data and simulations.
High field asymmetric waveform ion mobility spectrometry (FAIMS), also known as differential ion mobility spectrometry, is emerging as a tool for biomolecular analysis. In this article, the benefits and limitations of FAIMS for protein analysis are discussed. The principles and mechanisms of FAIMS separation of ions are described, and the differences between FAIMS and conventional ion mobility spectrometry are detailed. Protein analysis is considered from both the top-down (intact proteins) and the bottom-up (proteolytic peptides) perspective. The roles of FAIMS in the analysis of complex mixtures of multiple intact proteins and in the analysis of multiple conformers of a single protein are assessed. Similarly, the application of FAIMS in proteomics and targeted analysis of peptides are considered.
A dual-polarity linear ion trap (LIT) mass spectrometer was developed in this study, and the method for simultaneously controlling and detecting cations and anions was proposed and realized in the LIT. With the application of an additional dipolar DC field on the ejection electrodes of an LIT, dual-polarity mass spectra could be obtained, which include both the mass-to-charge (m/z) ratio and charge polarity information of an ion. Compared with conventional method, the ion ejection and detection efficiency could also be improved by about one-fold. Furthermore, ion–ion reactions within the LIT could be dynamically controlled and monitored by manipulating the distributions of ions with opposite charge polarities. This method was then used to control and study the reaction kinetics of ion–ion reactions, including electron transfer dissociation (ETD) and charge inversion reactions. A dual-polarity collision-induced dissociation (CID) experiment was proposed and performed to enhance the sequence coverage of a peptide ion. Ion trajectory simulations were also carried out for concept validation and system optimization.
We present a new two-plate linear ion trap mass spectrometer that overcomes both performance-based and miniaturization-related issues with prior designs. Borosilicate glass substrates are patterned with aluminum electrodes on one side and wire-bonded to printed circuit boards. Ions are trapped in the space between two such plates. Tapered ejection slits in each glass plate eliminate issues with charge build-up within the ejection slit and with blocking of ions that are ejected at off-nominal angles. The tapered slit allows miniaturization of the trap features (electrode size, slit width) needed for further reduction of trap size while allowing the use of substrates that are still thick enough to provide ruggedness during handling, assembly, and in-field applications. Plate spacing was optimized during operation using a motorized translation stage. A scan rate of 2300 Th/s with a sample mixture of toluene and deuterated toluene (D8) and xylenes (a mixture of o-, m-, p-) showed narrowest peak widths of 0.33 Th (FWHM).
Proof of concept evidence is presented for a new method for the determination of isoaspartate, an important post-translational modification. Chemical derivatization is performed using common reagents for the modification of carboxylic acids and shown to yield suitable diagnostic information with regard to isomerization at the aspartate residue. The diagnostic gas phase chemistry is probed by collision-induced dissociation mass spectrometry, on the timescale of the MS experiment and semi-quantitative calibration of the percentage of isoaspartate in a peptide sample is demonstrated.
Direct analysis of plant and animal tissue samples by laser electrospray mass spectrometry (LEMS) was investigated using low-energy, femtosecond duration laser vaporization at wavelengths of 800 and 1042 nm followed by nanospray postionization. Low-energy (<50 μJ), fiber-based 1042 nm LEMS (F-LEMS) allowed interrogation of the molecular species in fresh flower petal and leaf samples using 435 fs, 10 Hz bursts of 20 pulses from a Ytterbium-doped fiber laser and revealed comparable results to high energy (75–1120 μJ), 45 fs, 800 nm Ti:Sapphire-based LEMS (Ti:Sapphire-LEMS) measurements. Anthocyanins, sugars, and other metabolites were successfully detected and revealed the anticipated metabolite profile for the petal and leaf samples. Phospholipids, especially phosphatidylcholine, were identified from a fresh mouse brain section sample using Ti:Sapphire-LEMS without the application of matrix. These lipid features were suppressed in both the fiber-based and Ti:Sapphire-based LEMS measurements when the brain sample was prepared using the optimal cutting temperature compounds that are commonly used in animal tissue cryosections.
