Analysis Of Complex Biological Mixtures Research Articles

Botanical metabolite ions extraction from full electrospray ionization mass spectrometry using high-dimensional penalized regression.

Mass spectrometric data analysis of complex biological mixtures can be a challenge due to its vast datasets. There is lack of data treatment pipelines to analyze chemical signals versus noise. These tasks, so far, have been up to the discretion of the analysts. The aim of this work is to demonstrate an analytical workflow that would enhance the confidence in metabolomics before answering biological questions by serial dilution of botanical complex mixture and high-dimensional data analysis. Furthermore, we would like to provide an alternative approach to a univariate p-value cutoff from t-test for blank subtraction procedure between negative control and biological samples. A serial dilution of complex mixture analysis under electrospray ionization was proposed to study firsthand chemical complexity of metabolomics. Advanced statistical models using high-dimensional penalized regression were employed to study both the concentration and ion intensity relationship and the ion-ion relationship per second of retention time sub dataset. The multivariate analysis was carried out with a tool built in-house, so called metabolite ions extraction and visualization, which was implemented in R environment. A test case of the medicinal plant goldenseal (Hydrastis canandensis L.), showed an increase in metabolome coverage of features deemed as "important" by a multivariate analysis compared to features deemed as "significant" by a univariate t-test. For an illustration, the data analysis workflow suggested an unexpected putative compound, 20-hydroxyecdysone. This suggestion was confirmed with MS/MS acquisition and literature search. The multivariate analytical workflow selects "true" metabolite ions signals and provides an alternative approach to a univariate p-value cutoff from t-test, thus enhancing the data analysis process of metabolomics.

Effect of Fluorescent Labels on Peptide and Amino Acid Sample Dimensionality in Two Dimensional nLC × μFFE Separations.

Multidimensional separations present a unique opportunity for generating the high peak capacities necessary for the analysis of complex biological mixtures. We have coupled nano liquid chromatography with micro free flow electrophoresis (nLC × μFFE) to produce high peak capacity separations of peptide and amino acid mixtures. Currently, μFFE largely relies on laser-induced fluorescence (LIF) detection. We have demonstrated that the choice of fluorescent label significantly affects the fractional coverage and peak capacity of nLC × μFFE separations of peptides and amino acids. Of the labeling reagents assessed, Chromeo P503 performed the best for nLC × μFFE separations of peptides. A nLC × μFFE analysis of a Chromeo P503-labeled BSA tryptic digest produced a 2D separation that made effective use of the available separation space (48%), generating a corrected peak capacity of 521 in a 5 min separation window (104 peaks/min). nLC × μFFE separations of NBD-F-labeled peptides produced similar fractional coverage and peak capacity, but this reagent was able to react with multiple reaction sites, producing an unnecessarily complex analyte mixture. NBD-F performed the best for nLC × μFFE separations of amino acids. NBD-F-labeled amino acids produced a 2D separation that covered 36% of the available separation space, generating a corrected peak capacity of 95 in a 75 s separation window (76 peaks/min). Chromeo P503 and Alexa Fluor 488-labeled amino acids were not effectively separated in the μFFE dimension, giving 2D separations with poor fractional coverage and peak capacity.

The use of trimethylsilyl cyanide derivatization for robust and broad-spectrum high-throughput gas chromatography–mass spectrometry based metabolomics

Reproducible and quantitative gas chromatography-mass spectrometry (GC-MS)-based metabolomics analysis of complex biological mixtures requires robust and broad-spectrum derivatization. We have evaluated derivatization of complex metabolite mixtures using trimethylsilyl cyanide (TMSCN) and the most commonly used silylation reagent N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). For the comparative analysis, two metabolite mixtures, a standard complex mixture of 35 metabolites covering a range of amino acids, carbohydrates, small organic acids, phenolic acids, flavonoids and triterpenoids, and a phenolic extract of blueberry fruits were used. Four different derivatization methods, (1) direct silylation using TMSCN, (2) methoximation followed by TMSCN (M-TMSCN), (3) direct silylation using MSTFA, and (4) methoximation followed by MSTFA (M-MSTFA) were compared in terms of method sensitivity, repeatability, and derivatization reaction time. The derivatization methods were observed at 13 different derivatization times, 5 min to 60 h, for both metabolite mixtures. Fully automated sample derivatization and injection enabled excellent repeatability and precise method comparisons. At the optimal silylation times, peak intensities of 34 out of 35 metabolites of the standard mixture were up to five times higher using M-TMSCN compared with M-MSTFA. For direct silylation of the complex standard mixture, the TMSCN method was up to 54 times more sensitive than MSTFA. Similarly, all the metabolites detected from the blueberry extract showed up to 8.8 times higher intensities when derivatized using TMSCN than with MSTFA. Moreover, TMSCN-based silylation showed fewer artifact peaks, robust profiles, and higher reaction speed as compared with MSTFA. A method repeatability test revealed the following robustness of the four methods: TMSCN > M-TMSCN > M-MSTFA > MSTFA.

Selective 351 nm Photodissociation of Cysteine-Containing Peptides for Discrimination of Antigen-Binding Regions of IgG Fragments in Bottom-Up Liquid Chromatography–Tandem Mass Spectrometry Workflows

Despite tremendous inroads in the development of more sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies for mass spectrometry-based proteomics, there remains a significant need for enhancing the selectivity of MS/MS-based workflows for streamlined analysis of complex biological mixtures. Here, a novel LC-MS/MS platform based on 351 nm ultraviolet photodissociation (UVPD) is presented for the selective analysis of cysteine-peptide subsets in complex protein digests. Cysteine-selective UVPD is mediated through the site-specific conjugation of reduced cysteine residues with a 351 nm active chromogenic Alexa Fluor 350 (AF350) maleimide tag. Only peptides containing the AF350 chromophore undergo photodissociation into extensive arrays of b- and y-type fragment ions, thus providing a facile means for differentiating cysteine-peptide targets from convoluting peptide backgrounds. With the use of this approach in addition to strategic proteolysis, the selective analysis of diagnostic heavy-chain complementarity determining regions (CDRs) of single-chain antibody (scAb) fragments is demonstrated.

New cell separation technique for the isolation and analysis of cells from biological mixtures in forensic caseworks

AimTo isolate mucosal cells of the perpetrator in a sexual assault case from a complex mixture of his mucosal cells and the victim’s skin by micromanipulation prior to genomic analysis.MethodsTo capture and analyze mucosal cells we used the micromanipulation with on-chip low volume polymerase chain reaction (LV-PCR). Consensus DNA profiles were generated from 5 replicate experiments.Results and conclusionsWe validated the use of micromanipulation with on-chip LV-PCR for genomic analysis of complex biological mixtures in a fatal rape case. The perpetrator’s mucosal cells were captured from nipple swabs of the victim, and a single-source DNA profile was generated from cell mixtures. These data suggest that micromanipulation with on-chip LV-PCR is an effective forensic tool for the analysis of specific cells from complex samples.

Performance evaluation of long monolithic silica capillary columns in gradient liquid chromatography using peptide mixtures

A systematic study is reported on the performance of long monolithic capillary columns in gradient mode. Using a commercial nano-LC system, reversed-phase peptide separations obtained through UV-detection were conducted. The chromatographic performance, in terms of conditional peak capacity and peak productivity, was investigated for different gradient times (varying between 90 and 1320 min) and different column lengths (0.25, 1, 2 and 4 m) all originating from a single 4 m long column. Peak capacities reaching values up to n = 10 3 were measured in case of the 4 m long column demonstrating the high potential of these long monoliths for the analysis of complex biological mixtures, amongst others. In addition, it was found that the different column fragments displayed similar flow resistance as well as consistent chromatographic performance in accordance with chromatographic theory indicating that the chromatographic bed of the original 4 m long column possessed a structural homogeneity over its entire length.

CE separation of various analytes of biological origin using polyether ether ketone capillaries and contactless conductivity detection

The development of efficient and sensitive analytical methods for the separation, identification and quantification of complex biological samples is continuously a topic of high interest in biological science. In the present study, the possibility of using a polyether ether ketone (PEEK) capillary for the CE separation of peptides, proteins and other biological samples was examined. The performance of the tubing was compared with that of traditional silica capillaries. The CE analysis was performed using contactless conductivity detection (C(4)D), which eliminated any need for the detection window and was suitable for the detection of optically inactive compounds. In the PEEK capillary the cathodic EOF was low and of excellent stability even at extremes pH. In view of this fast biological anions were analyzed using an opposite end injection technique without compromising separation. A comparison of the performances of fused-silica and polymer capillaries during the separation of model sample mixtures demonstrated the efficiency and separation resolution of the latter to be higher and the reproducibility of the migration times and peak areas is better. Furthermore, PEEK capillaries allowed using simple experimental conditions without any complicated modification of the capillary surface or use of an intricate buffer composition. The PEEK capillaries are considered as an attractive alternative to the traditional fused-silica capillaries and may be used for the analysis of complex biological mixtures as well as for developing portable devices.

Controlled Protein Precipitation in Combination with Chip-Based Nanospray Infusion Mass Spectrometry. An Approach for Metabolomics Profiling of Plasma

Liquid chromatography-mass spectrometry (LC-MS) is a common method for profiling biological samples in metabolomics. However, LC-MS data of metabolomic studies are often affected by high noise levels, retention time shifts, and high variability in signal intensities. With a new chip-based nanoelectrospray source it becomes possible to directly infuse complex biological samples such as plasma without any chromatographic separation beforehand. In combination with highly diluted samples and long data acquisition times, the parallel analysis of hundreds of compounds is now possible. In a proof-of-concept study, 10 human plasma samples from females and males were analyzed with the intention to separate the two groups by their different metabolomes. The reproducibility was so high that statistical analysis of the data could be performed without prior normalization. Two groups of female and male samples were separated by a supervised machine learning algorithm, principal component analysis, and hierarchical clustering. Peaks contributing to the group separation were characterized by accurate mass measurement and MS-MS fragmentation and by spiking experiments. The feasibility of direct sample infusion using the new chip-based nanoelectrospray device opens a new dimension for the rapid parallel analysis of complex biological mixtures.

Capillary electrophoresis-electrospray ionization mass spectrometry of amino acids, peptides, and proteins.

Separation in capillary electrophoresis (CE) is based on the movement of charged compounds inside a background electrolyte under an applied potential. Because the mechanism of separation of CE differs from that of conventional high-performance liquid chromatography (HPLC), where separation is based on the analyte's hydrophobic properties, CE is often used as a complementary technique to HPLC. In addition, because CE is performed in narrow capillaries at atmospheric pressure, it is used as an alternative to HPLC, capable of handling small sample volumes while providing shorter analysis times with higher efficiency. For the analysis of amino acid, protein, and peptide mixtures in small volume samples such as in single cells, CE has rapidly evolved as a preferred separation technique. The combination of a high-efficiency separation technique, such as CE, with mass spectrometry (MS) detection provides a powerful system for the analysis of complex biological mixtures. In this chapter, a theoretical and practical approach to achieving high-performance CE-MS is discussed and the utility of CE-MS for the analysis of amino acids, peptides, and proteins is demonstrated.

Capillary electrophoresis mass spectrometry and its application to the analysis of biological mixtures.

Capillary electrophoresis (CE) mass spectrometry (MS), with its ability to separate compounds present in extremely small volume samples rapidly, with high separation efficiency, and with compound identification capability based on molecular weight, is an extremely valuable analytical technique for the analysis of complex biological mixtures. The highest sensitivities and separation efficiencies are usually achieved by using narrow capillaries (5-50 micro m i.d.) and by using sheathless CE-to-MS interfaces. The difficulties in CE-to-MS interfacing and the limited loadability of these narrow columns, however, have prevented CE-MS from becoming a widely used analytical technique. To remedy these limitations, several CE-MS interfacing techniques have recently been introduced. While electrospray ionization is the most commonly used ionization technique for interfacing CE-to-MS, matrix assisted laser desorption ionization has also been used, using both on-line and off-line techniques. Moreover, the high concentration detection limit of CE has been addressed by development of several sample concentration and sample focusing methods. In addition, a wide variety of techniques such as capillary zone electrophoresis, capillary isoelectric focusing, and on-column transient isotachophoresis have now been interfaced to MS. These advances have resulted in a rapid increase in the use of CE-MS in the analysis of complex biological mixtures. CE-MS has now been successfully applied to the analysis of a wide variety of compounds including amino acids, protein digests, protein mixtures, single cells, oligonucleotides, and various small molecules relevant to the pharmaceutical industry.

Tandem mass spectrometric analysis of complex biological mixtures

Proteomics is suddenly one of the hottest research areas of science, inspired by the completion of the sequencing of the human genome and of genomes of a number of other key species. The genome’s DNA sequence predicts those of its expressed proteins, and mass spectrometry (MS) is already playing a dominant role in the qualitative and quantitative analysis of these complex protein mixtures. Tandem mass spectrometry (MS/MS) for targeted compound analysis, pioneered by Cooks, is now a key technique for such problems, and this paper tries to show how it will be even more important in the future. The most used current MS approach to proteome analysis is applied after enzymatic degradation produces a complex mixture of peptides that provide MS or MS/MS sequence information. In the top down MS approach, ionization of protein mixtures yields mixtures of molecular ions that can be separated by MS for MS/MS sequence characterization of the selected “purified” protein. All posttranslational modifications and errors will be represented if MS/MS yields a complementary set of fragment ions, something not usually achieved with the smaller pieces of the bottom up approach. Here the new technique of electron capture dissociation (ECD) shows great promise, providing for more backbone cleavages than conventional methods and designating N- and C-terminal fragment ions. ECD specifically dissociates backbone bonds without appreciable loss of glycosylation, phosphorylation, or other posttranslational modifications, in contrast to energetic dissociation methods. This appears especially applicable for a proteome problem that involves quantitative determination of the extent of phosphorylation at two of 16 phosphorylation sites in the 50 kDa Lyn kinase.

A Method for Application of Samples to Matrix-Assisted Laser Desorption Ionization Time-of-Flight Targets That Enhances Peptide Detection

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry has become a fundamental tool for the identification and analysis of peptides and proteins. MALDI-TOF is well suited for the analysis of complex biological mixtures because samples are crystallized onto a solid support that can be washed to remove contaminants and salts prior to laser desorption. A number of approaches for immobilizing samples onto MALDI targets have been put forth. These include the use of different chemical matrices and the immobilization of samples onto different solid supports. In large part though, the preparation of MALDI targets has been an empirical exercise that often requires a unique series of conditions for every sample. Here, a simple method for the application of peptide mixtures onto MALDI targets is put forth. This method differs because peptides are added directly to a sample of nitrocellulose dissolved in acetone, allowing them to interact in solution-phase organic solvent. This solution-phase mixture is then spotted to the MALDI target and evaporated, forming a homogenous solid surface for laser desorption. This procedure is robust, highly sensitive, tolerant to detergents, and easily learned. In our hands, the method provides as much as a 10-fold enhancement to the detection of tryptic peptide fragments derived from in-gel digests.

Integrated approach to the multidimensional analysis of complex biological samples by microseparation techniques: Analysis of glycoprotein factor associated with cancer cachexia

Microanalytical separation techniques including capillary liquid chromatography, capillary electrophoresis and capillary electrochromatography are suitable for detection of diagnostically important changes in the metabolic profiles of biological fluids. A prototype instrument was employed to serve as an integrated platform for the analysis of urine sample from patients suffering from cancer cachexia. The instrument provides for convenient, rapid and efficient multidimensional approach towards method development which would facilitate simultaneous analysis of complex biological mixtures by the above techniques.

Mass spectrometric immunoassay.

A new, general method of immunoassay is demonstrated. The approach is based on the microscale immunoaffinity capture of target antigens followed by mass-specific identification and quantitation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Immunoaffinity capture of antigens effectively overcomes signal suppression effects typically encountered during traditional matrix-assisted laser desorption/ionization analysis of complex biological mixtures while simultaneously concentrating the analyte into a small volume. Mass spectrometric detection of antigens is unambiguous, as antigen signals are observed at characteristic mass-to-charge values in the mass spectrum, offering a high level of immunity to artifacts due to nonbiospecific retention of mixture components. However, the most important aspect of such mass-specific detection is the ability to use a single assay to screen biological systems for the presence of multiple, mass-resolved antigens. Analyte quantitation is possible by using a single antibody to capture both the antigen and an antigen variant which has been chemically modified to have a different mass. With proper calibration, the relative signal intensities of the two species in the mass spectrum can be used to determine the antigen concentration. Sample incubation and processing methods were such that a typical analysis could be performed in less than 1 h while subnanomolar sensitivities were maintained. The technique has been used for the rapid, selective, and quantitative screening of human blood for the presence of myotoxin a, and Mojave toxin form the venoms of the prairie rattlesnakes, Crotalus viridis viridis, and and the Mojave rattlesnake, Crotalus scutulatus scutulatus.

Characterization of ribonuclease B heterogeneity and the identification and removal of phosphate adducts by high resolution electrospray ionization Fourier transform ion clylotron resonance mass spectrometry

New instrumentation based on the combination of electrospray ionization (ESI) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been developed for the study of large biomolecules. The high resolution and accurate mass measurement possible with this instrumentation are demonstrated by application of the hetereogeneous glycoprotein, Ribonuclease B (RNase B). The high resolution routinely attainable allows unambiguous charge state assignments, and thus, precise mass determination for all ions observed and demonstrates the utility of ESI-FTICR for the analysis of complex biological mixtures. In addition, results are presented for the dissociation of RNase B in both the electrospray source and in the ICR cell. The results show that phosphate adducts to RNase B molecular ions are most readily dissociated in the heated capillary inlet, less effectively by collisional activation in the 1–10 Torr capillary-skimmer region, and with significantly reduced efficiency by collisional activation in the ICR cell, where other dissociation processes dominate. This trend is correlated with the extent of molecular ion solvation expected in the three regions, and suggests that phosphate adduct removal is most effective for solvated molecular ions.

Multiple scan modes in the hybrid tandem mass spectrometric screening and characterization of the glutathione conjugate of 2-furamide

The glutathione conjugate of 2-furamide has been screened for and structurally characterized by tandem mass spectrometry (MS/MS) by using a hybrid instrument of BEqQ design. Mass spectrometry experiments employed fast atom bombardment (FAB) ionization of a crude bile extract from a rat dosed with a 1:1 mixture of unlabeled and [ 13C 1]2-furamide. Initial screening for glutathione conjugates employed constant neutral loss scanning to detect the loss of 129 u, corresponding to the loss of the γ-glutamyl moiety of the conjugates. By direct comparison with control bile, [M + H] + ions of m/ z 417 and 418 were readily identified as candidate ions corresponding to the glutathione conjugates of unlabeled and 13C-labeled 2-furamide. Complementary screening information was generated by using a methylated bile extract, with constant neutral loss scanning to detect the loss of the methylated γ-glutamyl moiety (143 u). An alternative screening procedure employing parent ion scanning to detect the sodium adducts of methylated glutathione conjugates was also developed. Structural information was generated by first-generation product ion scanning of the protonated and sodium cationized forms of the candidate species, both native and derivatized. This provided a body of internally consistent evidence that the conjugate retains the pseudoaromatic furan ring system without ring hydroxylation. The utility of sequential mass spectrometry (MS/MS/MS) capability of the hybrid instrument in the analysis of complex biological mixtures was also demonstrated. Using the bile extract, first-generation product ions that formed in either the first or second field-free region of the double-focusing portion of the instrument were subsequently collisionally activated in the rf-only quadrupole followed by mass analysis of the second-generation product ions. Structural information so provided for the glutathione conjugate of 2-furamide further substantiated its retention of the pseudoaromatic furan ring system and facilitated plausible assignment of structures to ionic species generated through multiple decomposition events.