Pulsed Direct Current Arc-Induced Nanoelectrospray Ionization Mass Spectrometry.

This study explores the innovative field of pulsed direct current arc-induced nanoelectrospray ionization mass spectrometry (DCAI-nano-ESI-MS), which utilizes a low-temperature direct current (DC) arc to induce ESI during MS analyses. By employing a 15 kV output voltage, the DCAI-nano-ESI source effectively identifies various biological molecules, including angiotensin II, bradykinin, cytochrome C, and soybean lecithin, showcasing impressive analyte signals and facilitating multicharge MS in positive- and negative-ion modes. Notably, results show that the oxidation of fatty acids using a DC arc produces [M + O - H]- ions, which aid in identifying the location of C═C bonds in unsaturated fatty acids and distinguishing between isomers based on diagnostic ions observed during collision-induced dissociation tandem MS. This study presents an approach for identifying the sn-1 and sn-2 positions in phosphatidylcholine using phosphatidylcholine and nitrate adduct ions, accurately determining phosphatidylcholine molecular configurations via the Paternò-Büchi reaction. With all the advantages above, DCAI-nano-ESI holds significant promise for future analytical and bioanalytical applications.

The cold receptor TRPM8 activation leads to attenuation of endothelium-dependent cerebral vascular functions during head cooling.

Using the cranial window technique, we investigated acute effects of head cooling on cerebral vascular functions in newborn pigs. Head cooling lowered the rectal and extradural brain temperatures to 34.3 ± 0.6°C and 26.1 ± 0.6°C, respectively. During the 3-h hypothermia period, responses of pial arterioles to endothelium-dependent dilators bradykinin and glutamate were reduced, whereas the responses to hypercapnia and an endothelium-independent dilator sodium nitroprusside (SNP) remained intact. All vasodilator responses were restored after rewarming, suggesting that head cooling did not produce endothelial injury. We tested the hypothesis that the cold-sensitive TRPM8 channel is involved in attenuation of cerebrovascular functions. TRPM8 is immunodetected in cerebral vessels and in the brain parenchyma. During normothermia, the TRPM8 agonist icilin produced constriction of pial arterioles that was antagonized by the channel blocker AMTB. Icilin reduced dilation of pial arterioles to bradykinin and glutamate but not to hypercapnia and SNP, thus mimicking the effects of head cooling on vascular functions. AMTB counteracted the impairment of endothelium-dependent vasodilation caused by hypothermia or icilin. Overall, mild hypothermia produced by head cooling leads to acute reversible reduction of selected endothelium-dependent cerebral vasodilator functions via TRPM8 activation, whereas cerebral arteriolar smooth muscle functions are largely preserved.

Sensitive mass spectrometric determination of kinin-kallikrein system peptides in light of COVID-19.

The outbreak of COVID-19 has raised interest in the kinin-kallikrein system. Viral blockade of the angiotensin-converting enzyme 2 impedes degradation of the active kinin des-Arg(9)-bradykinin, which thus increasingly activates bradykinin receptors known to promote inflammation, cough, and edema-symptoms that are commonly observed in COVID-19. However, lean and reliable investigation of the postulated alterations is currently hindered by non-specific peptide adsorption, lacking sensitivity, and cross-reactivity of applicable assays. Here, an LC-MS/MS method was established to determine the following kinins in respiratory lavage fluids: kallidin, bradykinin, des-Arg(10)-kallidin, des-Arg(9)-bradykinin, bradykinin 1-7, bradykinin 2-9 and bradykinin 1-5. This method was fully validated according to regulatory bioanalytical guidelines of the European Medicine Agency and the US Food and Drug Administration and has a broad calibration curve range (up to a factor of 103), encompassing low quantification limits of 4.4-22.8 pg/mL (depending on the individual kinin). The application of the developed LC-MS/MS method to nasal lavage fluid allowed for the rapid (~ 2 h), comprehensive and low-volume (100 µL) determination of kinins. Hence, this novel assay may support current efforts to investigate the pathophysiology of COVID-19, but can also be extended to other diseases.

Heat-induced hyperthermia impacts the follicular fluid proteome of the periovulatory follicle in lactating dairy cows.

We hypothesized that heat-induced perturbations in cumulus cells surrounding the maturing oocyte may extend to the mural granulosa of the periovulatory follicle in the heat-stressed cow to subsequently the follicular fluid proteome. Lactating Holsteins were pharmacologically stimulated to have a dominant follicle that was capable of responding to a gonadotropin releasing hormone-induced luteinizing hormone surge. Following gonadotropin releasing hormone administration, cows were maintained at ~67 temperature humidity index (THI; thermoneutral conditions) or exposed to conditions simulating an acute heat stress event (71 to 86 THI; heat stress for ~12 h). Dominant follicle collection was conducted in the periovulatory period ~16 h after gonadotropin releasing hormone. Follicular fluid proteome from thermoneutral (n = 5) and hyperthermic (n = 5) cows was evaluated by quantitative tandem mass spectrometry (nano LC-MS/MS). We identified 35 differentially-abundant proteins. Functional annotation revealed numerous immune-related proteins. Subsequent efforts revealed an increase in levels of the proinflammatory mediator bradykinin in follicular fluid (P = 0.0456) but not in serum (P = 0.9319) of hyperthermic cows. Intrafollicular increases in transferrin (negative acute phase protein) in hyperthermic cows (P = 0.0181) coincided with a tendency for levels to be increased in the circulation (P = 0.0683). Nine out of 15 cytokines evaluated were detected in follicular fluid. Heat stress increased intrafollicular interleukin 6 levels (P = 0.0160). Whether hyperthermia-induced changes in the heat-stressed cow's follicular fluid milieu reflect changes in mural granulosa, cumulus, other cell types secretions, and/or transudative changes from circulation remains unclear. Regardless of origin, heat stress/hyperthermia related changes in the follicular fluid milieu may have an impact on components important for ovulation and competence of the cumulus-oocyte complex contained within the periovulatory follicle.

Extracting biomolecule collision cross sections from FT-ICR mass spectral line shape.

To extend the ion structure analysis capability of Fourier transform mass spectrometry (FT-MS), both time-domain and frequency-domain methods have been developed to extract ion collision cross sections (CCS) from high resolution mass spectra in Fourier transform ion cyclotron resonance (FT-ICR) cells. In this study, a new frequency-domain method, namely the line shape fitting method, was proposed to calculate ion CCSs from FT-ICR mass spectra line shape. Besides experimental data, simulated data with precisely controlled signal to noise levels and decay factors were also applied to characterize this method. Compared with the linewidth correction method previously proposed by our group, this line shape fitting method is more tolerant to noise, data length, and sampling rate, thus providing more consistent results. More importantly, CCS measurements of angiotensin I, bradykinin, ubiquitin and cytochrome c show that the resolving power is improved with the new method.

Ion Mobility-Mass Spectrometry Using a Dual-Gated 3D Printed Ion Mobility Spectrometer.

Described herein is the development of a 3D-printed drift-tube ion mobility spectrometer (IMS) which operates in the open air and is capable of being coupled to any mass spectrometer. The IMS possesses one electrospray focusing electrode, 31 drift electrodes with 7 mm inner diameters, and 2 ion gates at opposite ends of the IMS, totaling 109 mm in length. The second ion gate was timed with respect to the first ion gate to transmit portions of the separating ion packets to the MS at specified time intervals. By scanning the second ion gate and acquiring mass spectra during each time interval, we reconstructed ion mobility chronograms using mass spectra. Resolving powers of up to 45 were acquired using tetraalkylammonium cations. Separation is also demonstrated for solutions of amphetamines, opioids (fentanyls/fentanils), and bradykinin and angiotensin II. The highest mobility resolving powers were obtained when the injection times of the first and second ion gates were 0.3 and 1.0 ms, respectively. Experiments were performed on both an ion trap and triple quadruple mass analyzer to showcase the adaptability of the plastic IMS. Insights were gained into how ions separate in the open air compared to vacuum conditions with pure gas.

The blood fluke Schistosoma mansoni cleaves the coagulation protein high molecular weight kininogen (HK) but does not generate the vasodilator bradykinin.

BACKGROUND: Schistosomes are blood dwelling parasitic worms that cause the debilitating disease schistosomiasis. Here we examined the influence of the parasites on their external environment by monitoring the impact of adult Schistosoma mansoni worms on the murine plasma proteome in vitro and, in particular, on how the worms affect the blood coagulation protein high molecular weight kininogen (HK). METHODS: Following the incubation of adult schistosomes in murine plasma, two-dimensional differential in-gel electrophoresis (2D-DIGE) was conducted to look for changes in the plasma proteome compared with control plasma. A major change to the blood protein kininogen (HK) was observed, and the interaction of Schistosoma mansoni parasite with this protein alone was then investigated by western blot analysis and activity assays. Finally, the generation of bradykinin from HK was monitored using a bradykinin detection kit. RESULTS: The most striking change to the plasma proteome concerned HK; while the full-length protein was more abundant in control plasma, carboxyl-terminal truncated forms were more abundant in plasma that contained schistosomes. Incubating parasites in buffer with pure HK followed by Western blot analysis confirmed that human HK is degraded by the worms. The resulting digestion pattern differed from that brought about by kallikrein, a host serine protease that normally acts on HK to release the vasodilator bradykinin. We found that live schistosomes, while digesting HK, do not generate bradykinin nor do they cleave a chromogenic kallikrein substrate. Since the cleavage of HK by the worms is not impeded by the serine protease inhibitor PMSF but is blocked by the cysteine protease inhibitor E64c, we hypothesize that schistosome tegumental cysteine proteases are responsible for HK cleavage. CONCLUSIONS: Since proteomic and biochemical studies have revealed that the schistosome tegument contains two cysteine proteases belonging to the calpain family (SmCalp1 and SmCalp2) we conclude that these are likely responsible for the HK cleavage reported here. Schistosome cleavage of HK should help impede blood clotting and inflammation around the worms in vivo and so promote their ease of movement within the vasculature of their hosts.

Sodium dodecyl sulfate removal during electrospray ionization using cyclodextrins as simple sample solution additive for improved mass spectrometric detection of peptides.

Sodium dodecyl sulfate (SDS) removal is a vital procedure in SDS-assisted bottom-up proteomics because SDS affects the quality of the data in electrospray ionization mass spectrometry (ESI-MS). SDS removal methods provide efficient removal of SDS and improved peptide analysis, but would usually require time, specialised devices, and experienced analysts. Here, by simple addition of γ-cyclodextrin (γ-CD) to the solution at concentrations 1 to 2x the SDS in the sample, the SDS related signals in positive ionization ESI-MS can be significantly removed (70-99% reduction), without an additional sample manipulation step of extraction or purification. The mechanism for removal is based on the formation of tightly bound CD-SDS inclusion complexes, which hampered the generation of positively charged SDS multimers during ESI. For a sample with peptides (glu-val-phe, tyr-tyr-tyr, and bradykinin) and 3 mM SDS where 6 mM γ-CD was added, the %signal recoveries of peptides calculated by comparison with signals from standard samples without SDS were 49-59%. The space charge effect by SDS on bradykinin was also reduced, increasing the signal for bradykinin 12x in the presence of γ-CD. For a protein (bovine serum albumin, BSA) digest with 3 mM SDS, which is an expected concentration in trypsin treated samples, a noticeable 7-fold improvement in the peptide to SDS signal ratio and a 91% reduction of SDS signals were observed upon addition of 6 mM γ-CD. However, there were only small changes in the ESI-MS intensities for the BSA peptides (compared to without addition of γ-CD). This new approach to SDS signal removal using CDs in ESI-MS may find use in proteomic studies.

Electrokinetic Removal of Dodecyl Sulfate Micelles from Digested Protein Samples Prior to Electrospray-Ionization Mass Spectrometry.

In proteomics, dodecyl sulfate (DS-) as sodium salt is commonly used in protein solubilization prior to tryptic digestion, but the presence of the DS- hampers the electrospray ionization mass spectrometric (ESI-MS) analysis. The development of DS- depletion techniques is therefore important especially when dealing with small samples where there could be poor sensitivity due to sample loss or dilution during sample preparation. Here, we present a simple and fast electrokinetic removal method of DS- from small volumes of peptide and digested protein samples prior to ESI-MS. The selective removal was accomplished using an acidic extraction solution (ES) containing acetonitrile (ACN) inside a fused-silica capillary that was dipped into the sample. The use of acidic ES suppressed the electroosmotic flow; allowing the electrokinetic movement of DS- monomers and micelles into the capillary. The high amount of ACN present at the tip of the capillary served to collapse the micelles migrating into the capillary, thereby releasing the peptides that were bound to these micelles, facilitating peptide retention in the sample and efficient DS- removal. Increased % MS signal intensity (SI) restoration of the peptide was observed, while DS- removal was unaffected when the amount of ACN in the ES was increased. This is because of the micelle to solvent stacking mechanism (effective electrophoretic mobility reversal) working at high concentration of ACN for the improved recovery of the peptides. % MS SI restoration for the Z-Gly-Gly-Val and bradykinin peptides were 75-83% while % MS SI reduction of DS- was up to 99% under optimal conditions, that is, 40% ACN in the ES. Higher % peptide recoveries from digested protein samples were obtained using the proposed method compared to the conventional cold acetone precipitation method.

Theoretical and experimental comparison of one dimensional versus on-line comprehensive two dimensional liquid chromatography for optimized sub-hour separations of complex peptide samples.

This study was devoted to the search for conditions leading to highly efficient sub-hour separations of complex peptide samples with the objective of coupling to mass spectrometry. In this context, conditions for one dimensional reversed phase liquid chromatography (1D-RPLC) were optimized on the basis of a kinetic approach while conditions for on-line comprehensive two-dimensional liquid chromatography using reversed phase in both dimensions (on-line RPLCxRPLC) were optimized on the basis of a Pareto-optimal approach. Maximizing the peak capacity while minimizing the dilution factor for different analysis times (down to 5min) were the two objectives under consideration. For gradient times between 5 and 60min, 15cm was found to be the best column length in RPLC with sub-2µm particles under 800bar as system pressure. In RPLCxRPLC, for less than one hour as first dimension gradient time, the sampling rate was found to be a key parameter in addition to conventional parameters including column dimension, particle size, flow-rate and gradient conditions in both dimensions. It was shown that the optimum sampling rate was as low as one fraction per peak for very short gradient times (i.e. below 10min). The quality descriptors obtained under optimized RPLCxRPLC conditions were compared to those obtained under optimized RPLC conditions. Our experimental results for peptides, obtained with state of the art instrumentation, showed that RPLCxRPLC could outperform 1D-RPLC for gradient times longer than 5min. In 60min, the same peak intensity (same dilution) was observed with both techniques but with a 3-fold lower injected amount in RPLCxRPLC. A significant increase of the signal-to-noise ratio mainly due to a strong noise reduction was observed in RPLCxRPLC-MS compared to the one in 1D-RPLC-MS making RPLCxRPLC-MS a promising technique for peptide identification in complex matrices.

Achieving High Resolution Ion Mobility Separations Using Traveling Waves in Compact Multiturn Structures for Lossless Ion Manipulations.

We report on ion mobility (IM) separations achievable using traveling waves (TW) in a Structures for Lossless Ion Manipulations (SLIM) module having a 44 cm path length and 16 90° turns. The performance of the TW-SLIM module was evaluated for ion transmission and IM separations with different RF, TW parameters, and SLIM surface gaps in conjunction with mass spectrometry. In this work, TWs were created by the transient and dynamic application of DC potentials. The module demonstrated highly robust performance and, even with 16 closely spaced turns, achieving IM resolution performance and ion transmission comparable to a similar straight path module. We found an IM peak capacity of ∼31 and peak generation rate of 780 s(-1) for TW speeds of ∼80 m/s using the current multi-turn TW-SLIM module. The separations achieved for isomers of peptides and tetrasaccharides were found to be comparable to those from a ∼0.9-m drift tube-based IM-MS platform operated at the same pressure (4 Torr). The combined attributes of flexible design, low voltage requirements and lossless ion transmission through multiple turns for the present TW-SLIM module provides a basis for SLIM devices capable of achieving much greater IM resolution via greatly extended ion path lengths and using compact serpentine designs.

Use of deuterium labeling by high-temperature solid-state hydrogen-exchange reaction for mass spectrometric analysis of bradykinin biotransformation.

RATIONALE: Studies of molecular biodegradation by mass spectrometry often require synthetic compounds labeled with stable isotopes as internal standards. However, labeling is very expensive especially when a large number of compounds are needed for analysis of biotransformation. Here we describe an approach for qualitative and quantitative analysis using bradykinin (BK) and its in vitro degradation metabolites as an example. Its novelty lies in the use of deuterated peptides which are obtained by a high-temperature solid-state exchange (HSCIE) reaction. METHODS: Deuterated and native BK were analyzed by positive electrospray ionization high-resolution mass spectrometry (ESI-HRMS) using an Orbitrap Fusion mass spectrometer. High-energy collision-induced dissociation (HCD) experiments were performed on [M+H](+) and [M+2H](2+) ions in targeted-MS(2) mode with adjusted normalized HCD value. RESULTS: After the HSCIE reaction, each amino acid residue of the deuterated peptide contained deuterium atoms and the average degree of substitution was 5.5 atoms per the peptide molecule. The deuterated peptide demonstrated the same chromatographic mobility as the unlabeled counterpart, and lack of racemization during substitution with deuterium. Deuterium-labeled and unlabeled BKs were incubated with human plasma and their corresponding fragments BK(1-5) and BK(1-7), well known as the major metabolites, were detected. CONCLUSIONS: Quantitative assays demonstrated applicability of the heavy peptide for both sequencing and quantification of generated fragments. Applicability of the HSCIE deuterated peptide for analysis of routes of its degradation has been shown in in vitro experiments. Copyright © 2016 John Wiley & Sons, Ltd.

Bradykinin in Hemipepsis ustulata: A novel method for safely milking wasps.

Wasp venom characterization is of interest across multiple disciplines such as medicinal chemistry and evolutionary biology. A simple method is described herein to milk wasp venom without undue risks to the researcher. The wasps were immobilized by cooling for safe handling, restrained, and their venom was collected on parafilm. Bradykinin from Hemipepsis ustulata was identified by LC-MS/MS during method verification.

3D ToF-SIMS Analysis of Peptide Incorporation into MALDI Matrix Crystals with Sub-micrometer Resolution.

The analytical sensitivity in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is largely affected by the specific analyte-matrix interaction, in particular by the possible incorporation of the analytes into crystalline MALDI matrices. Here we used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to visualize the incorporation of three peptides with different hydrophobicities, bradykinin, Substance P, and vasopressin, into two classic MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA). For depth profiling, an Ar cluster ion beam was used to gradually sputter through the matrix crystals without causing significant degradation of matrix or biomolecules. A pulsed Bi3 ion cluster beam was used to image the lateral analyte distribution in the center of the sputter crater. Using this dual beam technique, the 3D distribution of the analytes and spatial segregation effects within the matrix crystals were imaged with sub-µm resolution. The technique could in the future enable matrix-enhanced (ME)-ToF-SIMS imaging of peptides in tissue slices at ultra-high resolution. Graphical Abstract ᅟ.

Synchronized Desorption Electrospray Ionization Mass Spectrometry Imaging.

Desorption electrospray ionization (DESI) has emerged as a powerful technique for mass spectral analysis and imaging under ambient conditions. Synchronization of DESI (sDESI) with the ion injection period (IT)of low-duty cycle mass spectrometers has been previously shown to improve sensitivity and reduce the amount of sample depleted during the acquisition of each spectrum (viz. MS scan time). In this report, we describe the development and characterization of an sDESI mass spectrometry imaging source (sDESI-MSI). Our results show that synchronization of DESI with the IT of an LTQ Orbitrap-XL mass spectrometer improves spatial resolution by factors of ∼4-6. In addition, under certain experimental conditions, synchronization was essential to acquire distinct MS images of low-intensity endogenous FAs (< 5% relative intensity) in fingermarks at high sampling frequencies (step sizes ≤ 75 µm). The magnitudes of these improvements in performance depend on the properties of the microdroplet spray, sample, and surface. Simulations that model analyte movement during desorption and the "washing effect" replicate the experimental results with the washing parameter having the greatest impact on performance. Thus, synchronization improves spatial resolution and sensitivity by decreasing the percentage of the total MS scan time that analytes are influenced by the "washing effect". Generally, synchronization of DESI with IT improves performance and expands the range of analytes, surfaces, and experimental conditions amenable to DESI-MSI, especially for analytes that are weakly attached to a surface.

Bradykinin and cysteinyl leukotriene concentrations in cell-salvaged blood before and after passage through negatively charged filters during clinical use in cancer patients: a pilot study.

It has been suggested that giving cell-salvaged blood through a leucocyte depletion filter can cause hypotension due to bradykinin released when factor XII and platelets are activated by the negatively charged surface of the filter. We measured the concentration of bradykinin and cysteinyl leukotrienes in cell-salvaged blood sampled before and after passage through a negatively charged leucodepletion filter in 24 consecutive patients with gynaecological or bowel cancer undergoing elective surgery with cell salvage. In no case was an increase in bradykinin concentration observed after passage through the filter; in 23 patients the post-filtration bradykinin concentration was zero (p = 0.007). The change in the concentration of cysteinyl leukotrienes detected during passage across the filter was not statistically significant (p = 0.1). Our findings do not support the suggestion that either bradykinin or cysteinyl leukotrienes are generated in cell-salvaged blood during passage through leucodepletion filters.

The quantitative impact of the mesopore size on the mass transfer mechanism of the new 1.9 µm fully porous Titan-C18 particles II--analysis of biomolecules.

The kinetic performances of 3.0 × 100 mm columns packed with 1.9 µm Titan-C18 particles with average mesopore sizes of 80 Å and 120 Å were investigated quantitatively for the analysis of biomolecules. Large mesopores are expected to speed up the rate of diffusivity of high-molecular-weight compounds across the stationary phase and to generate higher plate counts at high velocities. The mass transfer mechanism of bradykinin acetate salt (1060 Da) and insulin (5733 Da) was determined over a range of flow rates from 0.025 to 1.0 mL/min. The pore diffusivities of these two biomolecules were accurately measured from the peak parking method. Even though the gain in column efficiency was not found significant for small molecules such as valerophenone (162 Da), enlarging the average pore size from 80 to 120 Å induces a measurable diminution of the reduced plate height, h, of bradykinin (from 17 to 11 or -35% at a reduced velocity of 50) and a significant reduction for insulin (from 43 to 12 or -72% at a reduced velocity of 90). Remarkably, while the increase of the column efficiency for bradykinin is consistent with a faster diffusivity of bradykinin across the 120 Å Titan-C18 particles, the higher column efficiencies measured for insulin are mostly due to a faster absorption kinetics into the 120 Å than that into the 80 Å Titan-C18 particles. This result is supported by the fact that the effective pore diffusivity of insulin is even slightly smaller across the 120 Å than that across the 80 Å 1.9µm Titan-C18 particles.

Impact of excess gestational and post-weaning energy intake on vascular function of swine offspring.

BACKGROUND: The development of long-term vascular disease can be linked to the intrauterine environment, and maternal nutrition during gestation plays a critical role in the future vascular health of offspring. The purpose of this investigation was to test the hypothesis that a high-energy (HE) gestational diet, HE post-weaning diet, or their combination will lead to endothelial dysfunction in offspring. METHODS: Duroc × Landrace gilts (n = 16) were assigned to either a HE (10,144 Kcal/day, n = 8) or normal energy (NE: 6721 Kcal/day, n = 8) diet throughout pregnancy. Piglets were placed on either a NE or HE diet during the growth phase. At 3 months of age femoral arteries were harvested from offspring (n = 47). Endothelial-dependent and -independent vasorelaxation was measured utilizing wire-myography and increasing concentrations of bradykinin (BK) and sodium nitroprusside (SNP), respectively. RESULTS: BK and SNP induced vasorelaxation were significantly reduced in the femoral arteries of gestational HE offspring. However, no effect for the post-weaning diet on BK and SNP induced vasorelaxation was seen. This investigation demonstrates that a HE diet prenatally diminishes both BK and SNP induced vasorelaxation in swine. CONCLUSIONS: These findings suggest that a HE gestational diet can play a critical role in the development of offspring's vascular function, predisposing them to endothelial dysfunction. This dysfunction may lead to atherosclerotic disease development later in life.

Large-scale collision cross-section profiling on a traveling wave ion mobility mass spectrometer.

Ion mobility (IM) is a gas-phase electrophoretic method that separates ions according to charge and ion-neutral collision cross-section (CCS). Herein, we attempt to apply a traveling wave (TW) IM polyalanine calibration method to shotgun proteomics and create a large peptide CCS database. Mass spectrometry methods that utilize IM, such as HDMS(E), often use high transmission voltages for sensitive analysis. However, polyalanine calibration has only been demonstrated with low voltage transmission used to prevent gas-phase activation. If polyalanine ions change conformation under higher transmission voltages used for HDMS(E), the calibration may no longer be valid. Thus, we aimed to characterize the accuracy of calibration and CCS measurement under high transmission voltages on a TW IM instrument using the polyalanine calibration method and found that the additional error was not significant. We also evaluated the potential error introduced by liquid chromatography (LC)-HDMS(E) analysis, and found it to be insignificant as well, validating the calibration method. Finally, we demonstrated the utility of building a large-population peptide CCS database by investigating the effects of terminal lysine position, via LysC or LysN digestion, on the formation of two structural sub-families formed by triply charged ions.

On-plate self-desalting and matrix-free LDI MS analysis of peptides with a surface patterned sample support.

A hydrophobic-hydrophilic-hydrophobic pattern has been produced on the surface of a silicon substrate for selective enrichment, self-desalting, and matrix-free analysis of peptides in a single step. Upon sample application, the sample solution is first confined in a small area by a hydrophobic F-SAM outer area, after which salt contaminants and peptides are selectively enriched in the hydrophilic and hydrophobic areas, respectively. Simultaneously, matrix background noise is significantly reduced or eliminated because of immobilization of matrix molecules. As a result, the detection sensitivity is enhanced 20-fold compared with that obtained using the usual MALDI plate, and interference-free detection is achieved in the low m/z range. In addition, peptide ions can be identified unambiguously in the presence of NH4HCO3 (100 mM), urea (1 M), and NaCl (1 M). When the device was applied to the analysis of BSA digests, the peptide recovery and protein identification confidence were greatly improved.