Staggered multistep elution solid-phase extraction capillary electrophoresis/tandem mass spectrometry: a high-throughput approach in protein analysis

An approach based on staggered multistep elution solid-phase extraction (SPE) capillary electrophoresis/tandem mass spectrometry (CE/MS/MS) was developed in the analysis of digested protein mixtures. On-line coupling of SPE with CE/MS was achieved using a two-leveled two-cross polydimethylsiloxane (PDMS)-based interface. Multistep elution SPE was used prior to CE to provide an additional dimension of separation, thus extending the separation capacity for the peptide mixture analysis. By decreasing in the number of co-eluting peptides, problems stemming from ionization suppression and finite MS/MS duty cycle were reduced. As a result, sequence coverage increased significantly using multistep elution SPE-CE/MS/MS compared to one-step elution SPE-CE/MS/MS in the analysis of a single protein tryptic digest (49% vs. 18%) and a six protein tryptic digest (22-71% vs. 10-44%). A staggered CE method was incorporated to increase the throughput. The electropherograms of consecutive CE runs were partially overlapped by injecting the sample plug at a fixed time interval. With the use of a 5 min injection interval, slightly poor results were obtained in comparison with the sequential CE method while the total analysis time was reduced to 28%.     

A novel mass spectrometry cluster for high-throughput quantitative proteomics

We have developed and implemented a novel mass spectrometry (MS) platform combining the advantages of high mass accuracy and resolving power of Fourier transform ion cyclotron resonance (FTICR) with the economy and speed of multiple ion traps for tandem mass spectrometry. The instruments are integrated using novel algorithms and software and work in concert as one system. Using chromatographic time compression, a single expensive FTICR mass spectrometer can match the throughput of multiple relatively inexpensive ion trap instruments. Liquid chromatography (LC)-mass spectrometry data from the two types of spectrometers are aligned and combined to hybrid datasets, from which peptides are identified using accurate mass from the FTICR data and tandem mass spectra from the ion trap data. In addition, the high resolving power and dynamic range of a 12 tesla FTICR also allows precise label-free quantitation. Using two ion traps in parallel with one LC allows simultaneous MS/MS experiments and optimal application of collision induced dissociation and electrontransfer dissociation throughout the chromatographic separation for increased proteome coverage, characterization of post-translational modifications and/or simultaneous measurement in positive and negative ionization mode. An FTICR-ion trap cluster can achieve similar performance and sample throughput as multiple hybrid ion trap-FTICR instruments, but at a lower cost. We here describe the first such FTICR-ion trap cluster, its performance and the idea of chromatographic compression.     

Toward bioinspired galectin mimetics: identification of ligand-contacting peptides by proteolytic-excision mass spectrometry

Clinically relevant bioactivities of human galectins (adhesion/growth-regulatory galactoside-specific lectins) inspired the design of peptides as new tools to elicit favorable effects (e.g., in growth control) or block harmful binding (e.g., in tissue invasion). To obtain the bioinspired lead compounds, we combined a proteolytic fragmentation approach without/with ligand contact (excision) with mass spectrometric identification of affinity-bound protein fragments, using galectin-1 and -3 as models. Two peptides from the carbohydrate recognition domains were obtained in each case in experimental series rigorously controlled for specificity, and the [157-162] peptide of galectin-3 proved to be active in blocking lectin binding to a neoglycoprotein and to tumor cell surfaces. This approach affords peptide sequences for structural optimization and intrafamily/phylogenetic galectin comparison at the binding-site level with a minimal requirement of protein quantity, and it is even amenable to mixtures.     

Hardware-accelerated protein identification for mass spectrometry

An ongoing issue in mass spectrometry is the time it takes to search DNA sequences with MS/MS peptide fragments (see, e.g., Choudary et al., Proteomics 2001; 1: 651-667.) Search times are far longer than spectra acquisition time, and parallelization of search software on clusters requires doubling the size of a conventional computing cluster to cut the search time in half. Field programmable gate arrays (FPGAs) are used to create hardware-accelerated algorithms that reduce operating costs and improve search speed compared to large clusters. We present a novel hardware design that takes full spectra and computes 6-frame translation word searches on DNA databases at a rate of approximately 3 billion base pairs per second, with queries of up to 10 amino acids in length and arbitrary wildcard positions. Hardware post-processing identifies in silico tryptic peptides and scores them using a variety of techniques including mass frequency expected values. With faster FPGAs protein identifications from the human genome can be achieved in less than a second, and this makes it an ideal solution for a number of proteome-scale applications.     

High-throughput quantitative analysis by desorption electrospray ionization mass spectrometry

A newly developed high-throughput desorption electrospray ionization (DESI) source was characterized in terms of its performance in quantitative analysis. A 96-sample array, containing pharmaceuticals in various matrices, was analyzed in a single run with a total analysis time of 3 min. These solution-phase samples were examined from a hydrophobic PTFE ink printed on glass. The quantitative accuracy, precision, and limit of detection (LOD) were characterized. Chemical background-free samples of propranolol (PRN) with PRN-d₇ as internal standard (IS) and carbamazepine (CBZ) with CBZ-d₁₀ as IS were examined. So were two other sample sets consisting of PRN/PRN-d₇ at varying concentration in a biological milieu of 10% urine or porcine brain total lipid extract, total lipid concentration 250 ng/μL. The background-free samples, examined in a total analysis time of 1. 5 s/sample, showed good quantitative accuracy and precision, with a relative error (RE) and relative standard deviation (RSD) generally less than 3% and 5%, respectively. The samples in urine and the lipid extract required a longer analysis time (2. 5 s/sample) and showed RSD values of around 10% for the samples in urine and 4% for the lipid extract samples and RE values of less than 3% for both sets. The LOD for PRN and CBZ when analyzed without chemical background was 10 and 30 fmol, respectively. The LOD of PRN increased to 400 fmol analyzed in 10% urine, and 200 fmol when analyzed in the brain lipid extract.     

Ionic-liquid matrices for quantitative analysis by MALDI-TOF mass spectrometry

Ionic liquid matrices (ILMs) were tested as MALDI matrices for quantification of oligodeoxynucleotides (ODNs), peptides, and small proteins. Good calibrations with high linearity and reproducibility were achieved over a broad concentration range for all the tested ILMs in spite of their different physical states. However, the standard deviation is higher for ILMs that are solid with visible crystals. The experimental results indicate various ILMs have different sensitivity owing to changes in their cation components. More importantly, we found that the slopes of the calibration curves correlate with the inverse of the peptide molecular weights, presenting an opportunity to predict a priori, the relative sensitivities (slopes of calibration plots) for various analytes that have similar hydrophobicites.     

Multidimensional capillary array liquid chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for high-throughput proteomic analysis

A two-dimensional capillary array liquid chromatography system coupled with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) was developed for high-throughput comprehensive proteomic analysis, in which one strong cation-exchange (SCX) capillary chromatographic column was used as the first separation dimension and 10 parallel reversed-phase liquid chromatographic (RPLC) capillary columns were used as the second separation dimension. A novel multi-channel interface was designed and fabricated for on-line coupling of the SCX to RPLC column array system. Besides the high resolution based on the combination of SCX and RPLC separation, the developed new system provided the most rapid two-dimensional liquid chromatography (2D-LC) separation. Ten three-way micro-splitter valves used as stop-and-flow switches in transferring SCX fractions onto RPLC columns. In addition, the three-way valves also acted as mixing chambers of RPLC effluent with matrix. The system enables on-line mixing of the LC array effluents with matrix solution during the elution and directly depositing the analyte/matrix mixtures on MALDI plates from the tenplexed channels in parallel through an array of capillary tips. With the novel system, thousands of peptides were well separated and deposited on MALDI plates only in 150min for a complex proteome sample. Compared with common 2D-LC system, the parallel 2D-LC system showed about 10-times faster analytical procedure. In combination with a high throughput tandem time of flight mass spectrometry, the system was proven to be very effective for proteome analysis by analyzing a complicated sample, soluble proteins extracted from a liver cancer tissue, in which over 1202 proteins were identified.     

A centralized approach to tandem mass spectrometry method development for high-throughput ADME screening

A centralized approach to acquisition and dissemination of tandem mass spectrometry (MS/MS) conditions within an ADME-screening bioanalytical mass spectrometry group has been developed. The method development process uses two automated software products (Autoscan and Automaton) specifically designed for mass spectrometers manufactured by MDS Sciex. Both provide the ability to quickly determine selected reaction monitoring (SRM) transitions for hundreds of compounds per day. In addition, Autoscan determines optimal polarity and collision energy (CE). Automaton also determines the optimal declustering potential (DP) as well as the CE. The resulting optimized conditions are loaded into a central database for access by LC/MS/MS bioanalysis workstations in the group. The effect of DP and CE on the sensitivity was investigated. Optimization of DP improved signal response about 27% on average. For approximately 10% of compounds, signal enhancement was greater than 50% compared to the generic setting. A generic setting of DP = 25 V can be used for the majority of ADME-screening applications. Optimization of CE can have a much larger impact on signal intensity and a minimum of three CE settings should be tested. We have determined that CE values of 1, 30 and 45 V provide adequate coverage for most small molecule drug discovery analytes.     

Precise proteomic identification using mass spectrometry coupled with stable isotope labeling

Stable isotope labeling (SIL) can assist mass spectrometry to improve its specificity and throughput in protein identification, de novo sequencing, and characterization of post translational modifications. This Education article summarizes the unique characteristics of stable isotope-assisted mass spectrometry for accurate protein identification without requirements for ultrahigh mass accuracy. Some applications are discussed here to demonstrate the general experimental procedures, data interpretation, and the improvements in accuracy and throughput compared to the use of mass spectrometry alone.     

Toward automated chromatographic fingerprinting: A non-alignment approach to gas chromatography mass spectrometry data

In contrast to targeted analysis of volatile compounds, non-targeted approaches take information of known and unknown compounds into account, are inherently more comprehensive and give a more holistic representation of the sample composition. Although several non-targeted approaches have been developed, there's still a demand for automated data processing tools, especially for complex multi-way data such as chromatographic data obtained from multichannel detectors. This work was therefore aimed at developing a data processing procedure for gas chromatography mass spectrometry (GC–MS) data obtained from non-targeted analysis of volatile compounds. The developed approach uses basic matrix manipulation of segmented GC–MS chromatograms and PARAFAC multi-way modelling. The approach takes retention time shifts and peak shape deformations between samples into account and can be done with the freely available N-way toolbox for MATLAB. A demonstration of the new fingerprinting approach is presented using an artificial GC–MS data set and an experimental full-scan GC–MS data set obtained for a set of experimental wines.     

Selective identification and quantitative analysis of methionine containing peptides by charge derivatization and tandem mass spectrometry

To enable the development of a tandem mass spectrometry (MS/MS) based methodology for selective protein identification and differential quantitative analysis, a novel derivatization strategy is proposed, based on the formation of a "fixed-charge" sulfonium ion on the side-chain of a methionine amino acid residue contained within a protein or peptide of interest. The gas-phase fragmentation behavior of these side chain fixed charge sulfonium ion containing peptides is observed to result in exclusive loss of the derivatized side chain and the formation of a single characteristic product ion, independently of charge state or amino acid composition. Thus, fixed charge containing peptide ions may be selectively identified from complex mixtures, for example, by selective neutral loss scan mode MS/MS methods. Further structural interrogation of identified peptide ions may be achieved by subjecting the characteristic MS/MS product ion to multistage MS/MS (MS3) in a quadrupole ion trap mass spectrometer, or by energy resolved "pseudo" MS3 in a triple quadrupole mass spectrometer. The general principles underlying this fixed charge derivatization approach are demonstrated here by MS/MS, MS3 and "pseudo" MS3 analysis of side chain fixed-charge sulfonium ion derivatives of peptides containing methionine formed by reaction with phenacylbromide. Incorporation of "light" and "heavy" isotopically encoded labels into the fixed-charge derivatives facilitates the application of this method to the quantitative analysis of differential protein expression, via measurement of the relative abundances of the neutral loss product ions generated by dissociation of the light and heavy labeled peptide ions. This approach, termed "selective extraction of labeled entities by charge derivatization and tandem mass spectrometry" (SELECT), thereby offers the potential for significantly improved sensitivity and selectivity for the identification and quantitative analysis of peptides or proteins containing selected structural features, without requirement for extensive fractionation or otherwise enrichment from a complex mixture prior to analysis.     

On-tissue protein identification and imaging by MALDI-Ion mobility mass spectrometry

MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for the detection and localization of drugs, proteins, and lipids on-tissue. Nevertheless, this approach can only perform identification of low mass molecules as lipids, pharmaceuticals, and peptides. In this article, a combination of approaches for the detection and imaging of proteins and their identification directly on-tissue is described after tryptic digestion. Enzymatic digestion protocols for different kinds of tissues—formalin fixed paraffin embedded (FFPE) and frozen tissues—are combined with MALDI-ion mobility mass spectrometry (IM-MS). This combination enables localization and identification of proteins via their related digested peptides. In a number of cases, ion mobility separates isobaric ions that cannot be identified by conventional MALDI time-of-flight (TOF) mass spectrometry. The amount of detected peaks per measurement increases (versus conventional MALDI-TOF), which enables mass and time selected ion images and the identification of separated ions. These experiments demonstrate the feasibility of direct proteins identification by ion-mobility-TOF IMS from tissue. The tissue digestion combined with MALDI-IM-TOF-IMS approach allows a proteomics “bottom-up” strategy with different kinds of tissue samples, especially FFPE tissues conserved for a long time in hospital sample banks. The combination of IM with IMS marks the development of IMS approaches as real proteomic tools, which brings new perspectives to biological studies.     

Gas analysis by IMR-MS: a comparison to conventional mass spectrometry

Summary Ionisation of small molecules via ion-molecule reactions (IMR) offers the possibility to reduce fragmentation caused by high energy electron impact ionisation. Widely varying rate constants for the IMR processes allow to introduce additional species selectivity together with the mass selection in a quadrupole system. A comparison between low energy electron impact ionisation and IMR illustrates the potential of this new ionisation method in gas analysis applications.     

Probability-based protein identification by searching sequence databases using mass spectrometry data

Several algorithms have been described in the literature for protein identification by searching a sequence database using mass spectrometry data. In some approaches, the experimental data are peptide molecular weights from the digestion of a protein by an enzyme. Other approaches use tandem mass spectrometry (MS/MS) data from one or more peptides. Still others combine mass data with amino acid sequence data. We present results from a new computer program, Mascot, which integrates all three types of search. The scoring algorithm is probability based, which has a number of advantages: (i) A simple rule can be used to judge whether a result is significant or not. This is particularly useful in guarding against false positives. (ii) Scores can be compared with those from other types of search, such as sequence homology. (iii) Search parameters can be readily optimised by iteration. The strengths and limitations of probability-based scoring are discussed, particularly in the context of high throughput, fully automated protein identification.     

Effects of flow rate on high-throughput quantitative analysis of protein-precipitated plasma using liquid chromatography/tandem mass spectrometry

The effects of flow rate and column length on analyte response (peak area and height), total cycle time, column backpressure, and elution volume are presented. Rapid chromatographic separations and tandem mass spectrometric (MS/MS) detection are applied to the supernatant of protein-precipitated plasma standards containing four compounds from a drug discovery screen. The plasma samples were injected onto three C-18 columns (2 x 10,2.1 x 30 and 2.1 x 50 mm) at flow rates of 0.25, 0.50, 1.00 and 1.50 mL/min. The plasma samples were detected using a Sciex API 3000 tandem mass spectrometer operated in the Turbo Ionspray mode. A post-column split was used to maintain a flow rate of 0.25 mL/min into the mass spectrometer source to avoid differences in nebulization efficiency. The data show that diluted protein-precipitated plasma supernatants show average matrix effects (i.e. suppression) of 60.0% (2 x 10 mm), 89.3% (2 x 30 mm), and 76.7% (2 x 50 mm) of expected response at 10 ng/mL. Average matrix effects of 70.2% (2 x 10 mm), 88.9% (2 x 30 mm), and 81.2% (2 x 50 mm) of expected response at 1000 ng/mL plasma. The data also show if peak widths remain relatively constant, analytes are less sensitive as flow rates are increased. These data are consistent with the concentration-dependent relationship of ionspray in the range of flow rates studied. The data show that, while analyte response decreased proportionately to increases in flow rate, the analysis cycle times did not decrease proportionately.     

Flow-injection electrospray ionization mass spectrometry of crude cell extracts for high-throughput bacterial identification

Flow-injection electrospray ionization mass spectrometry (FI-ESI-MS) of unfractionated cell-free extracts obtained from bacterial cells suspended in a solvent mixture was investigated as a rapid analytical method for reproducible, high-throughput bacterial identification. Five bacterial strains (two Escherichia coli, two Bacillus spp. and one Brevibacillus laterosporus) were studied in this investigation. Axenically grown bacterial cells were suspended in an acidic organic solvent and the cell-free extract was sequentially injected into a solvent flow stream that was sprayed into the ionization chamber of the ESI-MS. The spectra produced contained reproducible information, which was useful for discriminating between the bacteria. Tandem mass spectrometry was used to characterize further the peaks, and at least three classes of macromolecules, namely phospholipids, glycolipids, and proteins, were found to contribute most to the spectral information. Bacterial extracts stored under different conditions gave very similar mass spectra for each of the five bacterial strains, indicating that the extracts were stable even at room temperature for up to 24 h, with no loss of information content, which has obvious implications for automated high-throughput analysis. An analysis of the components of the extracting solvent mixture and their effects on the spectral information showed that acetonitrile contributes most significantly to the extraction process and hence to the information content of the spectra.