A New Splitting Method for Both Analytical and Preparative LC/MS

This paper presents a novel splitting method for liquid chromatography/mass spectrometry (LC/MS) application, which allows fast MS detection of LC-separated analytes and subsequent online analyte collection. In this approach, a PEEK capillary tube with a micro-orifice drilled on the tube side wall is used to connect with LC column. A small portion of LC eluent emerging from the orifice can be directly ionized by desorption electrospray ionization (DESI) with negligible time delay (6~10 ms) while the remaining analytes exiting the tube outlet can be collected. The DESI-MS analysis of eluted compounds shows narrow peaks and high sensitivity because of the extremely small dead volume of the orifice used for LC eluent splitting (as low as 4 nL) and the freedom to choose favorable DESI spray solvent. In addition, online derivatization using reactive DESI is possible for supercharging proteins and for enhancing their signals without introducing extra dead volume. Unlike UV detector used in traditional preparative LC experiments, this method is applicable to compounds without chromophores (e.g., saccharides) due to the use of MS detector. Furthermore, this splitting method well suits monolithic column-based ultra-fast LC separation at a high elution flow rate of 4 mL/min.

Blotting Assisted by Heating and Solvent Extraction for DESI-MS Imaging

Imprints of potato sprout (Solanum tuberosum L.), gingko leaves (Gingko biloba L.) and strawberries (Fragaria x ananassa Duch.) were successfully imaged by desorption electrospray ionization mass spectrometry (DESI-MS) on TLC plates through blotting assisted by heating and/or solvent extraction. Ion images showing the distribution of significant compounds such as glycoalkaloid toxins in potato sprout, ginkgolic acids and flavonoids in ginkgo leaves, and sugars and anthocyanidin in strawberry were obtained. Practical implications of this work include analysis of a wide range of irregular or soft materials by different imprinting conditions without requiring the addition of matrices or use of specific kinds of surfaces.

Studying Reaction Intermediates Formed at Graphenic Surfaces

We report in-situ production and detection of intermediates at graphenic surfaces, especially during alcohol oxidation. Alcohol oxidation to acid occurs on graphene oxide-coated paper surface, driven by an electrical potential, in a paper spray mass spectrometry experiment. As paper spray ionization is a fast process and the time scale matches with the reaction time scale, we were able to detect the intermediate, acetal. This is the first observation of acetal formed in surface oxidation. The process is not limited to alcohols and the reaction has been extended to aldehydes, amines, phosphenes, sugars, etc., where reaction products were detected instantaneously. By combining surface reactions with ambient ionization and mass spectrometry, we show that new insights into chemical reactions become feasible. We suggest that several other chemical transformations may be studied this way. This work opens up a new pathway for different industrially and energetically important reactions using different metal catalysts and modified substrate.

Analysis of plasma protein adsorption onto DC-Chol-DOPE cationic liposomes by HPLC-CHIP coupled to a Q-TOF mass spectrometer

Plasma protein adsorption is regarded as a key factor in the in vivo organ distribution of intravenously administered drug carriers, and strongly depends on vector surface characteristics. The present study aimed to characterize the “protein corona” absorbed onto DC-Chol-DOPE cationic liposomes. This system was chosen because it is one of the most efficient and widely used non-viral formulations in vitro and a potential candidate for in vivo transfection of genetic material. After incubation of human plasma with cationic liposomes, nanoparticle–protein complex was separated from plasma by centrifugation. An integrated approach based on protein separation by one-dimensional 12% polyacrylamide gel electrophoresis followed by the automated HPLC-Chip technology coupled to a high-resolution mass spectrometer was employed for protein corona characterization. Thirty gel lanes, approximately 2 mm, were cut, digested and analyzed by HPLC-MS/MS. Fifty-eight human plasma proteins adsorbed onto DC-Chol-DOPE cationic liposomes were identified. The knowledge of the interactions of proteins with liposomes can be exploited for future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins in body fluids.

Coupling of Ultrafast LC with Mass Spectrometry by DESI

Recently we reported a desorption electrospray ionization (DESI) interface to combine liquid chromatography (LC) with mass spectrometry (MS) using a new LC eluent splitting strategy through a tiny orifice on LC capillary tube [J. Am. Soc. Mass Spectrom. 25, 286 (2014)]. The interface introduces negligible dead volume and back pressure, thereby allowing “near real-time” MS detection, fast LC elution, and online MS-directed purification. This study further evaluates the LC/DESI-MS performance with focus of using ultra-fast LC. Using a monolithic C₁₈ column, metabolites in urine can be separated within 1.6 min and can be online collected for subsequent structure elucidation (e.g., by NMR, UV, IR) in a recovery yield up to 99%. Using a spray solvent with alkaline pH, negative ions could be directly generated for acidic analytes (e.g., ibuprofen) in acidic LC eluent by DESI, offering a novel protocol to realize “wrong-way around” ionization for LC/MS analysis. In addition, DESI-MS is found to be compatible with ultra-performance liquid chromatography (UPLC) for the first time.      

Characterization of Rhodamine Self-Assembled Films Using Desorption Electrospray Ionization Mass Spectrometry

Growth process information and molecular structure identification are very important for characterization of self-assembled films. Here, we explore the possible application of desorption electrospray ionization mass spectrometry (DESI-MS) that provides the assembled information of rhodamine B (Rh B) and rhodamine 123 (Rh 123) films. With the help of lab-made DESI source, two characteristic ions [Rh B]⁺ and [Rh 123]⁺ are observed directly in the open environment. To evaluate the reliability of this technique, a comparative study of ultraviolet-visible (UV-vis) spectroscopy and our method is carried out, and the result shows good correlation. According to the signal intensity of characteristic ions, the layer-by-layer adsorption process of dyes can be monitored, and the thicknesses of multilayer films can also be comparatively determined. Combining the high sensitivity, selectivity, and speed of mass spectrometry, the selective adsorption of similar structure molecules under different pH is recognized easily from extracted ion chronograms. The variation trend of dyes signalling intensity with concentration of polyelectrolyte is studied as well, which reflects the effect of surface charge on dyes deposition. Additionally, the desorption area, surface morphology, and thicknesses of multilayer films are investigated using fluorescence microscope, scanning electron microscope (SEM), and atomic force microscopy (AFM), respectively. Because the desorption area was approximately as small as 2 mm², the distribution situation of organic dyes in an arbitrary position could be gained rapidly, which means DESI-MS has advantages on in situ analysis.

Simultaneous serum desalting and total protein determination by macroporous reversed-phase chromatography

Macroporous reversed-phase (mRP) chromatography was successfully used to develop an accurate and precise method for total protein in serum. The limits of detection (0.83 μg, LOD) and quantification (2.51 μg, LOQ) for the mRP method are comparable with those of the widely used micro BCA protein assay. The mRP method can be used to determine the total protein concentration across a wide dynamic range by detecting chromatographic peaks at 215 nm and 280 nm. The method has the added advantage of desalting and denaturing proteins, leading to more complete digestion by trypsin and to better LC–MS–MS identification in shotgun proteomics experiments.

Rapid enrichment of phosphopeptides by BaTiO3 nanoparticles after microwave-assisted tryptic digest of phosphoproteins, and their identification by MALDI-MS

We show that BaTiO₃ nanoparticles (NPs) can be used as a novel substrate for the rapid enrichment of phosphopeptides from microwave tryptic digests of α-casein and non-fat milk prior to their identification by MALDI-MS. Protein digestion is achieved by microwave tryptic digest for 50?s, and the resulting phosphopeptides can be effectively adsorbed on the surfaces of the NPs. The phosphopeptides were selectively detected via MALDI-MS. Digestion, enrichment and detection are accomplished within ～60?min. The method was applied to the indentification of 24 phosphopeptides from α-casein and of 21 phosphopeptides (of the α-casein type) from nonfat milk.

LC-MS/MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins

Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.

Measurement and Visualization of Mass Transport for the Flowing Atmospheric Pressure Afterglow (FAPA) Ambient Mass-Spectrometry Source

Ambient desorption/ionization mass spectrometry (ADI-MS) has developed into an important analytical field over the last 9 years. The ability to analyze samples under ambient conditions while retaining the sensitivity and specificity of mass spectrometry has led to numerous applications and a corresponding jump in the popularity of this field. Despite the great potential of ADI-MS, problems remain in the areas of ion identification and quantification. Difficulties with ion identification can be solved through modified instrumentation, including accurate-mass or MS/MS capabilities for analyte identification. More difficult problems include quantification because of the ambient nature of the sampling process. To characterize and improve sample volatilization, ionization, and introduction into the mass spectrometer interface, a method of visualizing mass transport into the mass spectrometer is needed. Schlieren imaging is a well-established technique that renders small changes in refractive index visible. Here, schlieren imaging was used to visualize helium flow from a plasma-based ADI-MS source into a mass spectrometer while ion signals were recorded. Optimal sample positions for melting-point capillary and transmission-mode (stainless steel mesh) introduction were found to be near (within 1 mm of) the mass spectrometer inlet. Additionally, the orientation of the sampled surface plays a significant role. More efficient mass transport resulted for analyte deposits directly facing the MS inlet. Different surfaces (glass slide and rough surface) were also examined; for both it was found that the optimal position is immediately beneath the MS inlet.

Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

Detection of follicular transport of lidocaine and metabolism in adipose tissue in pig ear skin by DESI mass spectrometry imaging

Desorption electrospray ionization (DESI) mass spectrometry imaging is demonstrated as a detection technique for penetration experiments of drugs in skin. Lidocaine ointment was used as the model compound in ex vivo experiments with whole pig ears as the skin model. Follicular transport of lidocaine into the deeper skin layers is demonstrated for the first time. Furthermore, metabolism of lidocaine to 3-OH-lidocaine was observed in subcutaneous tissue as well as in lobules of white adipose tissue surrounding the hair follicles. These results suggest that it is advantageous to use full thickness skin, including subcutaneous tissue, for skin metabolism studies.

Reactive desorption electrospray ionization mass spectrometry (DESI-MS) of natural products of a marine alga

Presented here is the optimization and development of a desorption electrospray ionization mass spectrometry (DESI-MS) method for detecting natural products on tissue surfaces. Bromophycolides are algal diterpene-benzoate macrolide natural products that have been shown to inhibit growth of the marine fungal pathogen Lindra thalassiae. As such, they have been implicated in antimicrobial chemical defense. However, the defense mechanisms are not yet completely understood. Precise detection of these compounds on algal tissue surfaces under ambient conditions without any disruptive sample processing could shed more light onto the processes involved in chemical defense of marine organisms. Conventional DESI-MS directly on algal tissue showed relatively low sensitivity for bromophycolide detection. Sensitivity was greatly improved by the addition of various anions including Cl⁻, Br⁻, and CF₃COO⁻ into the DESI spray solvent. Chloride adduction gave the highest sensitivity for all assayed anions. Density functional optimization of the bromophycolide anionic complexes produced during DESI supported this observation by showing that the chloride complex has the most favorable binding energy. Optimized DESI protocols allowed the direct and unambiguous detection of bromophycolides, including A, B, and E, from the surface of untreated algal tissue. Figure Desorption Electrospray Ionization, a novel technique for mass spectrometric analysis under open air conditions reveals the presence of naturally-occurring antibiotics on the surface of marine algae. Ab-initio calculations and experimental results indicate that sensitiviity could be greatly enhanced by means of dynamic complexation of these antibiotics with various small anions during the dynamic desorption process. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Rapid screening of active ingredients in drugs by mass spectrometry with low-temperature plasma probe

A high-throughput method for rapid screening of active ingredients in drugs has been developed with mass spectrometry coupled to a low-temperature plasma (LTP) probe ion source. Without sample preparation or pretreatment, the active ingredients of 11 types of commercial pharmaceuticals, including hormones, antipyretic analgesics, cardiovascular, digestant, neuro-psychotherapeutic, diuretic, antithyroid, sulfa anti-inflammatory, antiparastic, sedative-hypnotics, and antibacterial, were directly desorbed/ionized and detected by a linear ion trap mass spectrometry (MS). The structures of these ingredients were elucidated by tandem MS. The analysis of 18 methyltestosterone tablets could be accomplished within 1.9 min, which allows fast detection with a speed of approximate 600 samples within 1 h. This work demonstrated that LTP probe ion source combined with MS is a high-throughput method for screening of pharmaceuticals and potentially applied to on-line quality control in pharmaceutical industry.

Comparison and optimization of strategies for a more profound profiling of the sialylated N-glycoproteomics in human plasma using metal oxide enrichment

Glycosylation is an important posttranslational modification of proteins and plays a crucial role in both cellular functions and secretory pathways. Sialic acids (SAs), a family of nine-carbon-containing acidic monosaccharides, often terminate the glycan structures of cell surface molecules and secreted glycoproteins and perform an important role in many biological processes. Hence, a more profound profiling of the sialylated glycoproteomics may improve our knowledge of this modification and its effects on protein functions. Here, we systematically investigated different strategies to enrich the SA proteins in human plasma using a newly developed technology that utilizes titanium dioxide for sialylated N-glycoproteomics profiling by mass spectrometry. Our results showed that using a combination of a filter-aided sample preparation method, TiO₂ chromatography, multiple enzyme digestion, and two-dimensional reversed-phase peptide fractionation led to a more profound profiling of the SA proteome. In total, 982 glycosylation sites in 413 proteins were identified, among which 37.8 % were newly identified, to establish the largest database of sialic acid containing proteins from human plasma.

Amperometric sensing of hydrogen peroxide using a glassy cabon electode modified with silver nanoparticles on poly(alizarin yellow R)

A sensitive amperometric sensor for hydrogen peroxide (HP) was constructed that is based on a glassy carbon electrode (GCE) modified with silver nanoparticles on poly(alizarin yellow R). The polymer was electropolymerized onto the surface of the GCE by cyclic voltammetry (CV), and the AgNPs were then electrodeposited onto its surface. The electrode was characterized by scanning electron microscopy and CV, and used for amperometric determination of HP. The electrode exhibits a favorable catalytic activity towards the reduction of HP, with a linear response range from 1.0???M to 450???M and a detection limit of 0.32???M. The sensor also displays high selectivity, excellent reproducibility, and good long-term stability.

Using electrospray laser desorption ionization mass spectrometry to rapidly examine the integrity of proteins stored in various solutions

Electrospray laser desorption ionization mass spectrometry (ELDI/MS) allows the rapid desorption and ionization of proteins from solutions under ambient conditions. In this study, we have demonstrated the use of ELDI/MS to efficiently examine the integrity of the proteins stored in various solutions before they were further used for other biochemical tests. The protein standards were prepared in the solutions containing buffers, organic salts, inorganic salts, strong acid, strong base, and organic solvents, respectively, to simulate those collected from solvent extraction, filtration, dialysis, or chromatographic separation. Other than the deposit of a drop of the sample solution on the metallic sample plate in an ELDI source, no additional sample pretreatment is needed. The sample drop was then irradiated with a pulsed laser; this led to desorption of the analyte molecules, which subsequently entered the ESI plume to undergo post-ionization. Because adjustment of the composition of the sample solution is unnecessary, this technique appears to be useful for rapidly evaluating the integrity of proteins after storage or prior to further biochemical treatment. In addition, when using acid-free and low-organic-solvent ESI solutions for ELDI/MS analysis, the native conformations of the proteins in solution could be detected.

Enhanced Detection of Ubiquitin Isopeptides Using Reductive Methylation

Identification of ubiquitination (Ub) sites is of great interest due to the critical roles that the modification plays in cellular regulation. Current methods using mass spectrometry rely upon tryptic isopeptide diglycine tag generation followed by database searching. We present a novel approach to ubiquitin detection based upon the dimethyl labeling of isopeptide N-termini glycines. Ubiquitinated proteins were digested with trypsin and the resulting peptide mixture was derivatized using formaldehyde-D2 solution and sodium cyanoborohydride. The dimethylated peptide mixtures were next separated by liquid chromatography and analyzed on a quadrupole-TOF based mass spectrometer. Diagnostic b2′ and a1′ ions released from the isopeptide N-terminus upon collision-induced dissociation (CID) were used to spectrally improve the identification of ubiquitinated isopeptides. Proof of principle was established by application to a ubiquitinated protein tryptic digest spiked into a six-protein mix digest background. Extracted ion chromatograms of the a1′ and b2′ diagnostic product ions from the diglycine tag resulted in a significant reduction in signal complexity and demonstrated a selectivity towards the identification of diglycine branched isopeptides. The method was further shown to be capable of identifying diglycine isopeptides resulting from in-gel tryptic digests of ubiquitin enriched material from a His-Ub transfected cell line. We envisage that these ions may be utilized in global ubiquitination studies with post-acquisition MS/MS (or MSe) data interrogation on high resolution hybrid mass spectrometers.

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.

Surface molecular imprinting for chemiluminescence detection of the organophosphate pesticide chlorpyrifos

We report on a surface molecular imprinting strategy for synthesizing core-shell particles whose shell is imprinted with chlorpyrifos (CPF). The particles were prepared by copolymerization of the methacryloyl groups on the surface of silica particles modified with 3-methacryloxypropyl trimethoxysilane a functional monomer and a cross-linking agent. The imprinted particles exhibit larger binding capacity, faster binding kinetics, and higher recognition selectivity for CPF. Combined with highly sensitive chemiluminescence assay, the method was applied to the determination of CPF with a detection limit of 0.92?nM which is about 2 orders of magnitude lower than that by conventional CL method. The method also displays repeatability for more than 200 times.