A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics

A new matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometer with the novel "LIFT" technique (MALDI LIFT-TOF/TOF MS) is described. This instrument provides high sensitivity (attomole range) for peptide mass fingerprints (PMF). It is also possible to analyze fragment ions generated by any one of three different modes of dissociation: laser-induced dissociation (LID) and high-energy collision-induced dissociation (CID) as real MS/MS techniques and in-source decay in the reflector mode of the mass analyzer (reISD) as a pseudo-MS/MS technique. Fully automated operation including spot picking from 2D gels, in-gel digestion, sample preparation on MALDI plates with hydrophilic/hydrophobic spot profiles and spectrum acquisition/processing lead to an identification rate of 66% after the PMF was obtained. The workflow control software subsequently triggered automated acquisition of multiple MS/MS spectra. This information, combined with the PMF increased the identification rate to 77%, thus providing data that allowed protein modifications and sequence errors in the protein sequence database to be detected. The quality of the MS/MS data allowed for automated de novo sequencing and protein identification based on homology searching.     

Ideal velocity focusing in a reflectron time-of-flight mass spectrometer

A single-stage ion mirror in a time-of-flight (TOF) mass spectrometer (MS) can perform first order velocity focusing of ions initially located at a start focal plane while second order velocity focusing can be achieved using a double-stage reflectron. The situation is quite different when an ion source extraction field is taken into account. In this case which is common in any practical matrix-assisted laser desorption/ionization (MALDI) TOF-MS a single-stage reflectron, for example, cannot perform velocity focusing at all. In this paper an exact, analytic solution for an electric field inside a one-dimensional reflectron has been found to achieve universal temporal focusing of ions having an initial velocity distribution. The general solution is valid for arbitrary electric field distributions in the upstream (from the ion source to the reflectron) and downstream (from the reflectron to an ion detector) regions and in a decelerating part of the reflectron of a reflectron TOF mass spectrometer. The results obtained are especially useful for designing MALDI reflectron TOF mass spectrometers in which the initial velocity distribution of MALDI ions is the major limiting factor for achieving high mass resolution. Using analytical expressions obtained for an arbitrary case, convenient working formulas are derived for the case of a reflectron TOF-MS with a dual-stage extraction ion source. The special case of a MALDI reflectron TOF-MS with an ion source having a low acceleration voltage (or large extraction region) is considered. The formulas derived correct the effect of the acceleration regions in a MALDI ion source and after the reflectron before detecting ions.     

Increasing the mass resolution in a TOF aerosol mass spectrometer using a nonlinear post focusing method

The mass resolution for the time of flight aerosol mass spectrometer for aerosol component analysis is dependent on the initial direction and energy of the ions. We have found that the shape of the optimum post focusing electric field is nonlinear. The maximum electric potential should be applied to the ions whose initial direction is 90°. To determine the post focusing effects, a laser ablation mass spectrometer was installed. By using this LA-MS, it was found that the average energy distribution of the laser ablated ions was 8 eV. To establish an optimum mass resolution, a time delay and a high voltage are needed. The study results showed that 1500 ns and 3.7 kV, respectively, were the optimum parameters for time delay and voltage for this system. Using these optimized parameters, good resolution between the isotope mass signals of copper was achieved.     

Duty cycle enhancement of an orthogonal acceleration TOF mass spectrometer using an axially-resonant excitation linear ion trap

We report a new technique to enhance detection duty cycle of an orthogonal-acceleration time-of-flight mass spectrometer (oaTOF) over a broad mass range. To this end, we used an axially-resonant-excitation linear ion trap, which ejects ions axially and mass selectively into a non-mass-selective linear ion trap in front of the TOF pusher. A delay between the ejection timing of the non-mass-selective LIT and the push timing of the oaTOF was swept mass-synchronously with the axially-resonant-excitation linear ion trap, so that ions are detected with duty cycle larger than 60% over a wide mass range from m/z 174.1 to 1922.0, which is 3 to 10 times better than conventional oaTOF.     

Laser-induced electron impact ionization in a reflectron time-of-flight mass spectrometer

Some details of the generation of electrons by impinging a laser beam on a metal surface are described. It is shown that highly efficient electron generation is observed only during the laser pulse. Therefore, this technique delivers intense pulses of electrons. The process is investigated and different ion source set-ups are discussed. In conjunction with a time-of-flight mass spectrometer this technique can be used to produce mass spectra of different samples ranging from simple organic molecules to peptides.     

MALDI TOF/TOF tandem mass spectrometry as a new tool for amino acid analysis

This is the first report of an application of collisionally induced fragmentation of amino acids (AA) and their derivatives by MALDI TOF/TOF tandem mass spectrometry (MS). In this work, we collected the data on high-energy fragmentation reactions of a large group of protonated amino acids and their derivatives with the goal of determining which product ions are analyte specific and if yields of these fragment could be used for quantitative analysis. From 34 different amino acids (20 alpha-amino acids, beta-amino acids, homocysteine, GABA, and modified AA Met sulfone and sulfoxide, hydroxyproline, etc.) we observed that high yields of the target specific immonium ions and fragmentation patterns are most similar to EI or FAB CID on sector instruments. The major exceptions were two highly basic amino acids, Arg and Orn. It is noted that neither beta-, gamma-, nor delta-amino acids produce immonium ions. As might be predicted from high-energy CID work on peptides from the sectors and TOF/TOF, the presence of specific indicator ions in MALDI tandem MS allows distinguishing isomeric and isobaric amino acids. These indicator ions, in combination with careful control of data acquisition, ensure quantitative analysis of amino acids. We believe our data provide strong basis for the application of MALDI TOF/TOF MS/MS in qualitative and quantitative analysis of amino and organic acids, including application in clinical medicine.     

Investigation of an enhanced resolution triple quadrupole mass spectrometer for high-throughput liquid chromatography/tandem mass spectrometry assays

Triple quadrupole mass spectrometers, when operated in multiple reaction monitoring (MRM) mode, offer a unique combination of sensitivity, specificity, and dynamic range. Consequently, the triple quadrupole is the workhorse for high-throughput quantitation within the pharmaceutical industry. However, in the past, the unit mass resolution of quadrupole instruments has been a limitation when interference from matrix or metabolites cannot be eliminated. With recent advances in instrument design, triple quadrupole instruments now afford mass resolution of less than 0.1 Dalton (Da) full width at half maximum (FWHM). This paper describes the evaluation of an enhanced resolution triple quadrupole mass spectrometer for high-throughput bioanalysis with emphasis on comparison of selectivity, sensitivity, dynamic range, precision, accuracy, and stability under both unit mass (1 Da FWHM) and enhanced (相似文献   

Optimization of a MALDI TOF-TOF mass spectrometer for intact protein analysis

A MALDI TOF-TOF instrument was optimized and evaluated for intact protein analysis by tandem mass spectrometry. Ion source voltages and delay times were adjusted to affect an up to a 10-fold improvement in fragment ion yield compared to data obtained using default settings employed in peptide analysis. For large peptides (3-4.5 kDa), up to 90% of all possible b- and y-fragment ions were observed, which provides sufficient information for de novo sequencing and unambiguous protein identification. Product ion signals associated with preferential cleavages C-terminal to aspartic acid and glutamic acid residues and N-terminal to proline residues became dominant with increased protein molecular weight. Matrix effects were also evaluated and, among the eight matrices examined, alpha-cyano-4-hydroxycinnamic acid (CHCA) was found to produce the best intact protein tandem mass spectra for proteins up to 12 kDa. Optimized performance yielded detection limits of 50-125 fmol for proteins of 4 and 12 kDa, respectively. This improved performance has yielded an instrument with potential to be a useful tool in proteomic investigations via analysis of intact proteins.     

Pseudo-MS3 in a MALDI orthogonal quadrupole-time of flight mass spectrometer

Both the matrix selected and the laser fluence play important roles in MALDI-quadrupole/time of flight (QqTOF) fragmentation processes. "Hot" matrices, such as alpha-cyano4-hydroxycinnamic acid (HCCA), can increase fragmentation in MS spectra. Higher laser fluence also increases fragmentation. Typical peptide fragment ions observed in the QqTOF are a, b, and y ion series, which resemble low-energy CID product ions. This fragmentation may occur in the high-pressure region before the first mass-analyzing quadrupole. Fragment ions can be selected by the first quadrupole (Q1), and further sequenced by conventional MS/MS. This allows pseudo-MS3 experiments to be performed. For peptides of higher molecular weight, pseudo-MS3 can extend the mass range beyond what is usually accessible for sequencing, by allowing one to sequence a fragment ion of lower molecular weight instead of the full-length peptide. Peptides that predominantly show a single product ion after MS/MS yield improved sequence information when this technique is applied. This method was applied to the analysis of an in vitro phosphorylated peptide, where the intact enzymatically-generated peptide showed poor dissociation via MS/MS. Sequencing a fragment ion from the phosphopeptide enabled the phosphorylation site to be unambiguously determined.     

Selective ion isolation/rejection over a broad mass range in the quadrupole ion trap

Techniques are presented for mass-selective ion manipulation over a wide mass range in a three-dimensional quadrupole. The methods use an auxiliary, low-amplitude radio-frequency signal applied to the endcap electrodes. This signal is either held at a single frequency as the fundamental radio-frequency trapping amplitude is ramped or swept over a frequency range while the fundamental radio-frequency trapping amplitude is held at a fixed level. Ion isolation and ejection are demonstrated for ions formed within the ion trap using electron ionization and for ions injected into the ion trap formed either by an air-sustained glow discharge or by electrospray. Mass-selective ion ejection is used to reduce matrix-ion-induced space charge during ion injection, thereby producing signal enhancement for the detection of 2, 4, 6-trinitrotoluene in air. Mass-selective isolation of ions with mass-to-charge ratios above the normal operating range (m / z 650) for the ion trap is also demonstrated after injection of myoglobin ions formed via electrospray.     

Absolute Quantification of Rifampicin by MALDI Imaging Mass Spectrometry Using Multiple TOF/TOF Events in a Single Laser Shot

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the visualization of molecular distributions within tissue sections. While providing excellent molecular specificity and spatial information, absolute quantification by MALDI IMS remains challenging. Especially in the low molecular weight region of the spectrum, analysis is complicated by matrix interferences and ionization suppression. Though tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity and improve sensitivity by eliminating chemical noise, typical MALDI MS/MS modalities only scan for a single MS/MS event per laser shot. Herein, we describe TOF/TOF instrumentation that enables multiple fragmentation events to be performed in a single laser shot, allowing the intensity of the analyte to be referenced to the intensity of the internal standard in each laser shot while maintaining the benefits of MS/MS. This approach is illustrated by the quantitative analyses of rifampicin (RIF), an antibiotic used to treat tuberculosis, in pooled human plasma using rifapentine (RPT) as an internal standard. The results show greater than 4-fold improvements in relative standard deviation as well as improved coefficients of determination (R²) and accuracy (>93% quality controls, <9% relative errors). This technology is used as an imaging modality to measure absolute RIF concentrations in liver tissue from an animal dosed in vivo. Each microspot in the quantitative image measures the local RIF concentration in the tissue section, providing absolute pixel-to-pixel quantification from different tissue microenvironments. The average concentration determined by IMS is in agreement with the concentration determined by HPLC-MS/MS, showing a percent difference of 10.6%.

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Chip-based CE coupled to a quadrupole TOF mass spectrometer for the analysis of a glycopeptide

A novel approach using sheath flow chip-based CE coupled to a quadrupole TOF mass spectrometer was developed and applied to the analysis of an O-glycopeptide. The method provided good separation and sensitive analysis of the glycopeptide and its beta-elimination product. The beta-elimination reaction of the glycopeptide with ammonia or dimethylamine was adapted for changing the glycopeptide into deglycosylated form and for specifying the site of glycosylation. MS/MS analysis of the native peptide, glycopeptide and beta-elimination product enabled the identification of the glycosylation site through the comparison of the fragmentations and peptide sequence analysis.     

Comprehensive analysis of glycated human serum albumin tryptic peptides by off-line liquid chromatography followed by MALDI analysis on a time-of-flight/curved field reflectron tandem mass spectrometer

Glycated peptides arising from in vivo digestion of glycated proteins, usually called advanced glycation end (AGE) product peptides, are biologically relevant compounds due to their reactivity towards circulating and tissue proteins. To investigate their structures, in vitro glycation of human serum albumin (HSA) has been performed and followed by enzymatic digestion. Using different MALDI based approaches the digestion products obtained have been compared with those arising from enzymatic digestion of the protein. Results obtained using 2,5-dihydroxybenzoic acid (DHB) indicate this as the most effective matrix, leading to an increase in the coverage of the glycated protein. Off-line microbore liquid chromatography prior to MALDI analysis reveals that 63% of the free amino groups amenable to glycation are modified. In addition, the same approach shows the co-presence of underivatised peptides. This indicates that, regardless of the high glucose concentration employed for HSA incubation, glycation does not go to completion. Tandem mass spectrometric data suggest that the collision induced dissociation of singly charged glycated peptides leads to specific fragmentation pathways related to the condensed glucose molecule. The specific neutral losses derived from the activated glycated peptides can be used as signature for establishing the occurrence of glycation processes.     

A novel ion trap that enables high duty cycle and wide m/z range on an orthogonal injection TOF mass spectrometer

Although TOF analyzers with orthogonal ion injection provide the whole spectrum without scanning, their duty cycle is low compared with scanning analyzers in single ion monitoring mode. Typical duty cycle is in the range of 5% to 30% depending on the instrument geometry and ion m/z value. We present here a novel trapping/releasing setup, which offers the duty cycle near 100% over a wide range. Operation in the mass range from m/z 120 to almost 2000 is demonstrated. Ions are trapped in a short linear ion trap at the end of the collision cell in an axial pseudopotential well created by additional rf (“AC”) voltage applied to all four rods of the trap with the same amplitude and phase. The pseudopotential created by AC field is mass dependent, and by ramping down the AC voltage, ions can be released from the trap sequentially from high m/z to low, while all ions are gaining the same kinetic energy. Upon entering the TOF accelerator, ions with lower m/z catch up with heavier ions, and the AC ramp parameters can be selected to make all ions meet in the center of the TOF extraction region, resulting in sensitivity gains from 3 to 14 without loss of mass accuracy or resolution.     

The development of a high resolution mass spectrometer: A reminiscence

This article is a review of the events leading to the development of the double-focusing tandem mass spectrometer system, which is sometimes called the Johnson-Nier geometry. An essential feature of the geometry is that it eliminates second-order angular aberration, enhancing the sensitivity, without a loss in resolution. Flexibility in the choice of ion source and collector designs is assured because both are outside of regions of electric and magnetic fields. The vacuum housing permits high-temperature baking, ensuring ultrahigh vacuum conditions. Although introduced initially for the purpose of determining precise atomic masses, the design has found its greatest application in studies of structure of heavy molecules, making use of the high resolution to identify fragments. In many cases the composition of a molecule, or fragment, can be deduced from its exact mass by utilizing the known atomic masses of likely constituent atoms.     

On-line capillary liquid chromatography tandem mass spectrometry on an ion trap/ reflectron time-of-flight mass spectrometer using the sequence tag database search approach for peptide sequencing and protein identification

Capillary high-performance liquid chromatography has been coupled on-line with an ion trap storage/reflectron time-of-flight mass spectrometer to perform tandem mass spectrometry for tryptic peptides. Selection and fragmentation of the precursor ions were performed in a three-dimensional ion trap, and the resulting fragment ions were pulsed out of the trap into a reflectron time-of-flight mass spectrometer for mass analysis. The stored waveform inverse Fourier transform waveform was applied to perform ion selection and an improved tickle voltage optimization scheme was used to generate collision-induced dissociation. Tandem mass spectra of various doubly charged tryptic peptides were investigated where a conspicuous y ion series over a certain mass range defined a partial amino acid sequence. The partial sequence was used to determine the identity of the peptide or even the protein by database search using the sequence tag approach. Several peptides from tryptic digests of horse heart myoglobin and bovine cytochrome c were selected for tandem mass spectrometry (MS/MS) where it was demonstrated that the proteins could be identified based on sequence tags derived from MS/MS spectra. This approach was also utilized to identify protein spots from a two-dimensional gel separation of a human esophageal adenocarcinoma cell line.     

Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.     

Tryptamine: a Reactive Matrix for MALDI Mass Spectrometry

A possibility of using tryptamine as a reactive matrix for the analysis of non-polar carbonyl compounds by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has been shown. Presence of a terminal primary amine group in the tryptamine molecule predetermines the formation of Schiff bases from aliphatic and alicyclic carbonyl compounds. No additional matrix compounds are necessary to register MALDI mass spectra, because the excess of the derivatization agent plays the role of a matrix. MALDI mass spectra demonstrate high efficiency of desorption/ionization of the derivatives. To discover reactive matrices, a set of aromatic primary amines (mainly substituted anilines) has been tested, but they have not demonstrated matrix properties.     

An enhanced microfluidic chip coupled to an electrospray Qstar mass spectrometer for protein identification

The combination of microfabricated fluidic systems (muFAB) and electrospray mass spectrometers (ESI-MS) will provide multiplexed and integrated analytical systems for proteins and other biomolecules. Implementation of this novel approach requires the development of robust and user-friendly muFAB devices. Here, we present new approaches that improve the robustness, user friendliness and performance of muFAB devices coupled to MS. First, we present the development of a convenient mount to connect a muFAB device to the ESI-MS and the incorporation of filters in the reservoirs and exit of the muFAB. This mount facilitates interfacing and significantly reduces the chemical noise observed by the MS. Furthermore, we demonstrate improvements in sample handling and delivery by using either a nonaqueous electrolyte or a cationic coating on the surfaces in the muFAB device and transfer capillary. These improvements are applied to protein analysis by continuous infusion of proteolytic digests.