Linkage Determination of Linear Oligosaccharides by MSn (n > 2) Collision-Induced Dissociation of Z1 Ions in the Negative Ion Mode

Obtaining unambiguous linkage information between sugars in oligosaccharides is an important step in their detailed structural analysis. An approach is described that provides greater confidence in linkage determination for linear oligosaccharides based on multiple-stage tandem mass spectrometry (MSⁿ, n >2) and collision-induced dissociation (CID) of Z₁ ions in the negative ion mode. Under low energy CID conditions, disaccharides ¹⁸O-labeled on the reducing carbonyl group gave rise to Z₁ product ions (m/z 163) derived from the reducing sugar, which could be mass-discriminated from other possible structural isomers having m/z 161. MS³ CID of these m/z 163 ions showed distinct fragmentation fingerprints corresponding to the linkage types and largely unaffected by sugar unit identities or their anomeric configurations. This unique property allowed standard CID spectra of Z₁ ions to be generated from a small set of disaccharide samples that were representative of many other possible isomeric structures. With the use of MSⁿ CID (n = 3 – 5), model linear oligosaccharides were dissociated into overlapping disaccharide structures, which were subsequently fragmented to form their corresponding Z₁ ions. CID data of these Z₁ ions were collected and compared with the standard database of Z₁ ion CID using spectra similarity scores for linkage determination. As the proof-of-principle tests demonstrated, we achieved correct determination of individual linkage types along with their locations within two trisaccharides and a pentasaccharide.

Interpretation and Deconvolution of Nanodisc Native Mass Spectra

Nanodiscs are a promising system for studying gas-phase and solution complexes of membrane proteins and lipids. We previously demonstrated that native electrospray ionization allows mass spectral analysis of intact Nanodisc complexes at single lipid resolution. This report details an improved theoretical framework for interpreting and deconvoluting native mass spectra of Nanodisc lipoprotein complexes. In addition to the intrinsic lipid count and charge distributions, Nanodisc mass spectra are significantly shaped by constructive overlap of adjacent charge states at integer multiples of the lipid mass. We describe the mathematical basis for this effect and develop a probability-based algorithm to deconvolute the underlying mass and charge distributions. The probability-based deconvolution algorithm is applied to a series of dimyristoylphosphatidylcholine Nanodisc native mass spectra and used to provide a quantitative picture of the lipid loss in gas-phase fragmentation.

Spectral Reproducibility and Quantification of Peptides in MALDI of Samples Prepared by Micro-Spotting

Previously, we reported that MALDI spectra of peptides became reproducible when temperature was kept constant. Linear calibration curves derived from such spectral data could be used for quantification. Homogeneity of samples was one of the requirements. Among the three popular matrices used in peptide MALDI [i.e., α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and sinapinic acid (SA)], homogeneous samples could be prepared by conventional means only for CHCA. In this work, we showed that sample preparation by micro-spotting improved the homogeneity for all three cases.

A colorimetric probe for the rapid and selective determination of mercury(II) based on the disassembly of gold nanorods

We report on a novel mercury(II)-controlled approach for the disassembly of gold nanorods (AuNRs) that has led to a detection system for Hg(II). The modified AuNRs were fabricated by functionalizing AuNRs with L-cysteine via a thiol group chemisorption-type of interaction. L-cysteine induces the assembly of AuNRs through cooperative electrostatic interaction upon which the color of the solution of the AuNRs changes from blue-green to gray dark. The addition of Hg(II), in turn, causes the disassembly of the modified AuNRs and the color of the solution returns to blue-green. This effect enables the optical determination of Hg(II) in aqueous solution, with a linear response in the 0.5 to 250 μM Hg(II) concentration range, an excellent selectivity for Hg(II), and with recoveries ranging from 99 % to 106 % in spiked environmental water samples.

Efficient Methods to Generate Reproducible Mass Spectra in Matrix-Assisted Laser Desorption Ionization of Peptides

In our previous matrix-assisted laser desorption ionization (MALDI) studies of peptides, we found that their mass spectra were virtually determined by the effective temperature in the early matrix plume, T_early, when samples were rather homogeneous. This empirical rule allowed acquisition of quantitatively reproducible spectra. A difficulty in utilizing this rule was the complicated spectral treatment needed to get T_early. In this work, we found another empirical rule that the total number of particles hitting the detector, or TIC, was a good measure of the spectral temperature and, hence, selection of spectra with the same TIC resulted in reproducible spectra. We also succeeded in obtaining reproducible spectra throughout a measurement by controlling TIC near a preset value through feedback adjustment of laser pulse energy. Both TIC selection and TIC control substantially reduced the shot-to-shot spectral variation in a spot, spot-to-spot variation in a sample, and even sample-to-sample variation in MALDI using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid as matrix. Based on the utilization of acquired data, TIC control was more efficient than TIC selection by an order of magnitude. Both techniques produced calibration curves with excellent linearity, suggesting their utility in quantification of peptides.

Spectral signatures for the classification of microbial species using Raman spectra

In general, classification-based methods based on confocal Raman microscopy are focused on targeted studies under which the spectral libraries are collected under controlled instrument parameters, which facilitate analyses via standard multivariate data analysis methods and cross-validation. We develop and compare approaches to transform spectra collected at different spectral ranges and varying levels of resolution into a single lower-dimension spectral signature library. This will result in a more robust analysis method able to accommodate spectra accumulated at different times and conditions. We demonstrate these approaches on a relevant test case; the identification of microbial species from a natural environment. The training data were based on samples prepared for three unique species collected at two time points and the test data consisted of blinded unknowns prepared and analyzed at a later date with different instrument parameters. The results indicate that using reduced dimension representations of the spectral signatures improves classification accuracy over basic alignment protocols. In particular, utilizing the microbial species partial least squares discriminant analysis classifier on the blinded samples based on alignment achieved ~78 % accuracy, while both binning and peak selection approaches yielded 100 % accuracy.

Assignment of the Stereochemistry and Anomeric Configuration of Sugars within Oligosaccharides Via Overlapping Disaccharide Ladders Using MSn

A systematic approach is described that can pinpoint the stereo-structures (sugar identity, anomeric configuration, and location) of individual sugar units within linear oligosaccharides. Using a highly modified mass spectrometer, dissociation of linear oligosaccharides in the gas phase was optimized along multiple-stage tandem dissociation pathways (MSⁿ, n = 4 or 5). The instrument was a hybrid triple quadrupole/linear ion trap mass spectrometer capable of high-efficiency bidirectional ion transfer between quadrupole arrays. Different types of collision-induced dissociation (CID), either on-resonance ion trap or beam-type CID could be utilized at any given stage of dissociation, enabling either glycosidic bond cleavages or cross-ring cleavages to be maximized when wanted. The approach first involves optimizing the isolation of disaccharide units as an ordered set of overlapping substructures via glycosidic bond cleavages during early stages of MSⁿ, with explicit intent to minimize cross-ring cleavages. Subsequently, cross-ring cleavages were optimized for individual disaccharides to yield key diagnostic product ions (m/z 221). Finally, fingerprint patterns that establish stereochemistry and anomeric configuration were obtained from the diagnostic ions via CID. Model linear oligosaccharides were derivatized at the reducing end, allowing overlapping ladders of disaccharides to be isolated from MSⁿ. High confidence stereo-structural determination was achieved by matching MSⁿ CID of the diagnostic ions to synthetic standards via a spectral matching algorithm. Using this MSⁿ (n = 4 or 5) approach, the stereo-structures, anomeric configurations, and locations of three individual sugar units within two pentasaccharides were successfully determined.

Isolating stem cells in the inter-follicular epidermis employing synchrotron radiation-based Fourier-transform infrared microspectroscopy and focal plane array imaging

Normal function and physiology of the epidermis is maintained by the regenerative capacity of this tissue via adult stem cells (SCs). However, definitive identifying markers for SCs remain elusive. Infrared (IR) spectroscopy exploits the ability of cellular biomolecules to absorb in the mid-IR region (λ?=?2.5–25?μm), detecting vibrational transitions of chemical bonds. In this study, we exploited the cell’s inherent biochemical composition to discriminate SCs of the inter-follicular skin epidermis based on IR-derived markers. Paraffin-embedded samples of human scalp skin (n?=?4) were obtained, and 10-μm thick sections were mounted for IR spectroscopy. Samples were interrogated in transmission mode using synchrotron radiation-based Fourier-transform IR (FTIR) microspectroscopy (15?×?15?μm) and also imaged employing globar-source FTIR focal plane array (FPA) imaging (5.4?×?5.4?μm). Dependent on the location of derived spectra, wavenumber–absorbance/intensity relationships were examined using unsupervised principal component analysis. This approach showed clear separation and spectral differences dependent on cell type. Spectral biomarkers concurrently associated with segregation of SCs, transit-amplifying cells and terminally-differentiated cells of epidermis were primarily PO ₂ ^? vibrational modes (1,225 and 1,080?cm^?1), related to DNA conformational alterations. FPA imaging coupled with hierarchical cluster analysis also indicated the presence of specific basal layer cells potentially originating from the follicular bulge, suggested by co-clustering of spectra. This study highlights PO ₂ ^? vibrational modes as potential putative SC markers.

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

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

Structural Distinction of Diacyl-, Alkylacyl,and Alk-1-Enylacyl Glycerophosphocholines as [M – 15]– Ions by Multiple-Stage Linear Ion-Trap Mass Spectrometry with Electrospray Ionization

We describe a linear ion-trap (LIT) multiple-stage (MSⁿ) mass spectrometric approach towards differentiation of alkylacyl, alk-1-enylacyl- and diacyl-glycerophoscholines (PCs) as the [M – 15]^– ions desorbed by electrospray ionization (ESI) in the negative-ion mode. The MS⁴ mass spectra of the [M – 15 – R₂′CH = CO]^– ions originated from the three PC subfamilies are readily distinguishable, resulting in unambiguous distinction of the lipid classes. This method is applied to two alkyl ether rich PC mixtures isolated from murine bone marrow neutrophils and kidney, respectively, to explore its utility in the characterization of complex PC mixture of biological origin, resulting in the realization of the detailed structures of the PC species, including various classes and many minor isobaric isomers.

Highly sensitive determination of doxorubicin and daunorubicin based on their effect on the resonance light scattering signals of the ethidium-DNA complex

We have developed a resonance light scattering (RLS) quenching assay for the highly sensitive determination of doxorubicin (DOX) and daunorubicin (DAU). It is based on the reduction of the intensity of the shoulder of the RLS spectra at 443?nm. The intensity of the RLS of the ethidium-DNA system decrease linearly on addition of trace quantities of DOX or DAU within the concentration range of 0.008 to 12.0???g?mL^?1 for DOX, and of 0.010 to 21.0???g?mL^?1 for DAU. The detection limits are 3.0 and 5.0?ng?mL^?1, respectively. The assay was successfully applied to the determination of DAU in synthetic and serum samples. Compared to the reported methods for anthracyclines, this assay displays higher sensitivity, lower detection limits, and a wider linear range.

Soft Ionization of Saturated Hydrocarbons, Alcohols and Nonpolar Compounds by Negative-Ion Direct Analysis in Real-Time Mass Spectrometry

Large polarizable n-alkanes (approximately C18 and larger), alcohols, and other nonpolar compounds can be detected as negative ions when sample solutions are injected directly into the sampling orifice of the atmospheric pressure interface of the time-of-flight mass spectrometer with the direct analysis in real time (DART) ion source operating in negative-ion mode. The mass spectra are dominated by peaks corresponding to [M + O₂]￣^?. No fragmentation is observed, making this a very soft ionization technique for samples that are otherwise difficult to analyze by DART. Detection limits for cholesterol were determined to be in the low nanogram range.

Bioactivity fingerprint analysis of cyclooxygenase-2 ligands from radix Aconiti by ultrafiltration–UPLC–MSn

A novel fingerprinting method, bioactivity fingerprint analysis, based on an ultrafiltration–ultraperformance liquid chromatography–multistage tandem mass spectrometry (UPLC–MS ⁿ ) method is proposed for the quality control of herbal medicines from the bioactivity viewpoint concerning the efficacy of herbal medicines. The bioactivity fingerprints reflecting the anti-inflammatory activities of radix Aconiti and radix Aconiti preparata were established. With use of ultrafiltration UPLC–MS ⁿ , 11 cyclooxygenase-2 ligands from radix Aconiti preparata and 14 cyclooxygenase-2 ligands from radix Aconiti were found after incubation with cyclooxygenase-2. Twelve of the cyclooxygenase-2 ligands were identified by the ultraperformance UPLC–MS ⁿ method. The enrichment factor of each peak in the bioactivity fingerprint was calculated and was demonstrated to be characteristic, which makes bioactivity fingerprint analysis for the quality control of herbal medicines possible from the viewpoint of their bioactivities.

Sensitive detection of trace hemoglobin using fluorescence method based on functionalized quantum dots

The fluorescence quenching of quantum dots by hemoglobin has been demonstrated to depend on surface functionalization, and this property has been utilized to construct a novel fluorescent method for rapid, sensitive, and selective detection of trace hemoglobin in urine at microgram level. This method shows low interference and high selectivity for hemoglobin with a limit of detection of 4.3 μg L^?1 in water and 66.1 μg L^?1 in urine, which are lower than those of currently used methods in labs and clinics. Spike and recovery tests in raw, acidified, and alkalized urine samples exhibit good recovery rates for the spiked concentrations close to the limit of detection.

Novel chemiluminescent assay for staphylococcal enterotoxin B

An enzyme-linked immunosorbent assay, a horseradish peroxidase-catalyzed fluorogenic reaction, and chemiluminescence (CL) analysis have been combined to develop a sandwich ELISA for Staphylococcal enterotoxin B (SEB) using monoclonal antibodies for different epitopes of SEB. The enzyme catalyzed reaction of 3-(4-hydroxyphenyl propionate) with the urea complex of hydrogen peroxide produced a fluorescent dimer which was detected by chemiluminescence analysis. The CL response to SEB is linear in the range from 6.0 to 564?pg?mL^?1 (r?=?0.9993), and the detection limit is 3.3?pg?mL^?1 (S/N?=?3). Intra- and interassay coefficients of variation are <7.0% at three concentrations (24, 96 and 384?pg?mL^?1). The method was applied to the analysis of SEB in serum, lake water and milk samples. The results compared well with those obtained by conventional ELISAs.

Non-Direct Sequence Ions in the Tandem Mass Spectrometry of Protonated Peptide Amides—an Energy-Resolved Study

The fragmentation reactions of the MH⁺ ions of Leu-enkephalin amide and a variety of heptapeptide amides have been studied in detail as a function of collision energy using a QqToF beam type mass spectrometer. The initial fragmentation of the protonated amides involves primarily formation of b_n ions, including significant loss of NH₃ from the MH⁺ ions. Further fragmentation of these b_n ions occurs following macrocyclization/ring opening leading in many cases to b_n ions with permuted sequences and, thus, to formation of non-direct sequence ions. The importance of these non-direct sequence ions increases markedly with increasing collision energy, making peptide sequence determination difficult, if not impossible, at higher collision energies.

Characterization of the alkaloids in goldenseal (Hydrastis canadensis) root by high resolution Orbitrap LC-MSn

Liquid chromatography coupled to multistage mass spectrometry (LC-MSⁿ) is being used increasingly in pharmaceutical research and for quality control in herbal medicines because of its superior sensitivity and selectivity. In this study, a rapid, high-resolution liquid chromatography-mass spectrometry (LC-MSⁿ) method was developed to separate and identify alkaloids in the root extract of goldenseal, which is one of the 20 most popular herbal supplements used worldwide. In total, 28 alkaloids were separated and characterized including one novel compound and 21 identified, or tentatively identified, for the first time in goldenseal. The current high-resolution LC-MSⁿ method provides a rapid and definitive means of profiling the composition of goldenseal root and will provide a useful tool in understanding the bioactivity of this medicinal plant.

Charge Site Mass Spectra: Conformation-Sensitive Components of the Electron Capture Dissociation Spectrum of a Protein

A conventional electron capture dissociation (ECD) spectrum of a protein is uniquely characteristic of the first dimension of its linear structure. This sequence information is indicated by summing the primary c ^m+ and z ^m+? products of cleavage at each of its molecular ion’s inter-residue bonds. For example, the ECD spectra of ubiquitin (M?+?nH)ⁿ⁺ ions, n?=?7–13, provide sequence characterization of 72 of its 75 cleavage sites from 1843 ions in seven c ^(1–7)+ and eight z ^(1–8)+? spectra and their respective complements. Now we find that each of these c/z spectra is itself composed of “charge site (CS)” spectra, the c ^m+ or z ^m+? products of electron capture at a specific protonated basic residue. This charge site has been H-bonded to multiple other residues, producing multiple precursor ion forms; ECD at these residues yields the multiple products of that CS spectrum. Closely similar CS spectra are often formed from a range of charge states of ubiquitin and KIX ions; this indicates a common secondary conformation, but not the conventional α-helicity postulated previously. CS spectra should provide new capabilities for comparing regional conformations of gaseous protein ions and delineating ECD fragmentation pathways.

Characterization and analysis of mycobacteria and Gram-negative bacteria and co-culture mixtures by Raman microspectroscopy, FTIR, and atomic force microscopy

The molecular composition of mycobacteria and Gram-negative bacteria cell walls is structurally different. In this work, Raman microspectroscopy was applied to discriminate mycobacteria and Gram-negative bacteria by assessing specific characteristic spectral features. Analysis of Raman spectra indicated that mycobacteria and Gram-negative bacteria exhibit different spectral patterns under our experimental conditions due to their different biochemical components. Fourier transform infrared (FTIR) spectroscopy, as a supplementary vibrational spectroscopy, was also applied to analyze the biochemical composition of the representative bacterial strains. As for co-cultured bacterial mixtures, the distribution of individual cell types was obtained by quantitative analysis of Raman and FTIR spectral images and the spectral contribution from each cell type was distinguished by direct classical least squares analysis. Coupled atomic force microscopy (AFM) and Raman microspectroscopy realized simultaneous measurements of topography and spectral images for the same sampled surface. This work demonstrated the feasibility of utilizing a combined Raman microspectroscopy, FTIR, and AFM techniques to effectively characterize spectroscopic fingerprints from bacterial Gram types and mixtures.