Automatic internal calibration in liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry of protein digests

Accurately measured peptide masses can be used for large-scale protein identification from bacterial whole-cell digests as an alternative to tandem mass spectrometry (MS/MS) provided mass measurement errors of a few parts-per-million (ppm) are obtained. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) routinely achieves such mass accuracy either with internal calibration or by regulating the charge in the analyzer cell. We have developed a novel and automated method for internal calibration of liquid chromatography (LC)/FTICR data from whole-cell digests using peptides in the sample identified by concurrent MS/MS together with ambient polydimethyl-cyclosiloxanes as internal calibrants in the mass spectra. The method reduced mass measurement error from 4.3 +/- 3.7 ppm to 0.3 +/- 2.3 ppm in an E. coli LC/FTICR dataset of 1000 MS and MS/MS spectra and is applicable to all analyses of complex protein digests by FTICRMS. Copyright (c) 2006 John Wiley & Sons, Ltd.

Multiplexed quantitative proteomics using differential metabolic 15N-labeling

There are several advantages of using metabolic labeling in quantitative proteomics. The early pooling of samples compared to post-labeling methods eliminates errors from different sample processing, protein extraction and enzymatic digestion. Metabolic labeling is also highly efficient and relatively inexpensive compared to commercial labeling reagents. However, methods for multiplexed quantitation in the MS-domain (or ‘non-isobaric’ methods), suffer from signal dilution at higher degrees of multiplexing, as the MS/MS signal for peptide identification is lower given the same amount of peptide loaded onto the column or injected into the mass spectrometer. This may partly be overcome by mixing the samples at non-uniform ratios, for instance by increasing the fraction of unlabeled proteins. We have developed an algorithm for arbitrary degrees of nonisobaric multiplexing for relative protein abundance measurements. We have used metabolic labeling with different levels of 15N, but the algorithm is in principle applicable to any isotope or combination of isotopes. Ion trap mass spectrometers are fast and suitable for LC-MS/MS and peptide identification. However, they cannot resolve overlapping isotopic envelopes from different peptides, which makes them less suitable for MS-based quantitation. Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry is less suitable for LC-MS/MS, but provides the resolving power required to resolve overlapping isotopic envelopes. We therefore combined ion trap LC-MS/MS for peptide identification with FTICR LC-MS for quantitation using chromatographic alignment. We applied the method in a heat shock study in a plant model system (A. thaliana) and compared the results with gene expression data from similar experiments in literature.

Comparison of bottom-up protein identification methods

With the rapid development of proteomics, a number of different methods appeared for the basic task of protein identification. We made a simple comparison between a common liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow using an ion trap mass spectrometer and a combined LC-MS and LC-MS/MS method using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and accurate peptide masses. To compare the two methods for protein identification, we grew and extracted proteins from E. coli using established protocols. Cystines were reduced and alkylated, and proteins digested by trypsin. The resulting peptide mixtures were separated by reversed-phase liquid chromatography using a 4 h gradient from 0 to 50% acetonitrile over a C18 reversed-phase column. The LC separation was coupled on-line to either a Bruker Esquire HCT ion trap or a Bruker 7 tesla APEX-Qe Qh-FTICR hybrid mass spectrometer. Data-dependent Qh-FTICR-MS/MS spectra were acquired using the quadrupole mass filter and collisionally induced dissociation into the external hexapole trap. Proteins were in both schemes identified by Mascot MS/MS ion searches and the peptides identified from these proteins in the FTICR MS/MS data were used for automatic internal calibration of the FTICR-MS data, together with ambient polydimethylcyclosiloxane ions.

Analysis of chain and blood group type and branching pattern of sialylated oligosaccharides by negative ion electrospray tandem mass spectrometry

We previously reported sequence determination of neutral oligosaccharides by negative ion electrospray tandem mass spectrometry on a quadrupole-orthogonal time-of-flight instrument with high sensitivity and without the need of derivatization. In the present report, we extend our strategies to sialylated oligosaccharides for analysis of chain and blood group types together with branching patterns. A main feature in the negative ion mass spectrometry approach is the unique double glycosidic cleavage induced by 3-glycosidic substitution, producing characteristic D-type fragments which can be used to distinguish the type 1 and type 2 chains, the blood group related Lewis determinants, 3,6-disubstituted core branching patterns, and to assign the structural details of each of the branches. Twenty mono- and disialylated linear and branched oligosaccharides were used for the investigation, and the sensitivity achieved is in the femtomole range. To demonstrate the efficacy of the strategy, we have determined a novel complex disialylated and monofucosylated tridecasaccharide that is based on the lacto-N-decaose core. The structure and sequence assignment was corroborated by :methylation analysis and H-1 NMR spectroscopy.

Liquid ultraviolet matrix-assisted laser desorption/ionization - mass spectrometry for automated proteomic analysis

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet -- matrix-assisted laser desorption/ionisation -- mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. The low-femtomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydroxybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and low-mass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.

Liquid ultraviolet matrix-assisted laser desorption/ionization mass spectrometry for automated proteomic analysis

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet - matrix-assisted laser desorption/ ionisation - mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. U. Am. Soc. Mass Spectrom. 1998, 9, 166-174). The low-ferntomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydrox-ybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and lowmass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.

Use of the statistical properties of the wavelet-transform coefficients for optimization of integration time in Fourier transform spectrometry

We show that an analysis of the mean and variance of discrete wavelet coefficients of coaveraged time-domain interferograms can be used as a specification for determining when to stop coaveraging. We also show that, if a prediction model built in the wavelet domain is used to determine the composition of unknown samples, a stopping criterion for the coaveraging process can be developed with respect to the uncertainty tolerated in the prediction.

Liquid AP-UV-MALDI enables stable ion yields of multiply charged peptide and protein ions for sensitive analysis by mass spectrometry

In biological mass spectrometry (MS), two ionization techniques are predominantly employed for the analysis of larger biomolecules, such as polypeptides. These are nano-electrospray ionization [1, 2] (nanoESI) and matrix-assisted laser desorption/ionization [3, 4] (MALDI). Both techniques are considered to be “soft”, allowing the desorption and ionization of intact molecular analyte species and thus their successful mass-spectrometric analysis. One of the main differences between these two ionization techniques lies in their ability to produce multiply charged ions. MALDI typically generates singly charged peptide ions whereas nanoESI easily provides multiply charged ions, even for peptides as low as 1000 Da in mass. The production of highly charged ions is desirable as this allows the use of mass analyzers, such as ion traps (including orbitraps) and hybrid quadrupole instruments, which typically offer only a limited m/z range (< 2000–4000). It also enables more informative fragmentation spectra using techniques such as collisioninduced dissociation (CID) and electron capture/transfer dissociation (ECD/ETD) in combination with tandem MS (MS/MS). [5, 6] Thus, there is a clear advantage of using ESI in research areas where peptide sequencing, or in general, the structural elucidation of biomolecules by MS/MS is required. Nonetheless, MALDI with its higher tolerance to contaminants and additives, ease-of-operation, potential for highspeed and automated sample preparation and analysis as well as its MS imaging capabilities makes it an ionization technique that can cover bioanalytical areas for which ESI is less suitable. [7, 8] If these strengths could be combined with the analytical power of multiply charged ions, new instrumental configurations and large-scale proteomic analyses based on MALDI MS(/MS) would become feasible.

Liquid matrix deposition on conductive hydrophobic surfaces for tuning and quantitation in UV-MALDI mass spectrometry

With its highly fluctuating ion production matrix-assisted laser desorption/ionization (MALDI) poses many practical challenges for its application in mass spectrometry. Instrument tuning and quantitative ion abundance measurements using ion signal alone depend on a stable ion beam. Liquid MALDI matrices have been shown to be a promising alternative to the commonly used solid matrices. Their application in areas where a stable ion current is essential has been discussed but only limited data have been provided to demonstrate their practical use and advantages in the formation of stable MALDI ion beams. In this article we present experimental data showing high MALDI ion beam stability over more than two orders of magnitude at high analytical sensitivity (low femtomole amount prepared) for quantitative peptide abundance measurements and instrument tuning in a MALDI Q-TOF mass spectrometer. Samples were deposited on an inexpensive conductive hydrophobic surface and shrunk to droplets <10 nL in size. By using a sample droplet <10 nL it was possible to acquire data from a single irradiated spot for roughly 10,000 shots with little variation in ion signal intensity at a laser repetition rate of 5-20 Hz.

Liquid matrices for analyses by UV-MALDI mass spectrometry

Data are presented for a pH-adjustable liquid UV-matrix-assisted laser desorption ionization (MALDI) matrix for mass spectrometry analysis. The liquid matrix system possesses high analytical sensitivity within the same order of magnitude as that achievable by the commonly used solid UV-MALDI matrices such as 2,5-dihydroxybenzoic acid but with improved spot homogeneity and reproducibility. The pH of the matrix has been adjusted by the addition of up to 0.35% trifluoroacetic acid and up to 200 mM ammonium bicarbonate, achieving an on-target pH range of 3.5-8.6. Alteration of the pH does not seem to affect the overall sample signal intensity or signal-to-noise ratio achievable, nor does it affect the individual peptide ion signals from a mixture of peptides with varying isoelectric points (p1). In addition, the pH adjustment has allowed for the performance of a tryptic digest within the diluted pH-optimized liquid matrix.

Evaluation of peak-picking algorithms for protein mass spectrometry

Peak picking is an early key step in MS data analysis. We compare three commonly used approaches to peak picking and discuss their merits by means of statistical analysis. Methods investigated encompass signal-to-noise ratio, continuous wavelet transform, and a correlation-based approach using a Gaussian template. Functionality of the three methods is illustrated and discussed in a practical context using a mass spectral data set created with MALDI-TOF technology. Sensitivity and specificity are investigated using a manually defined reference set of peaks. As an additional criterion, the robustness of the three methods is assessed by a perturbation analysis and illustrated using ROC curves.

Assessment of the consistency of near-infrared water vapor line intensities using high-spectral-resolution ground-based Fourier transform measurements of solar radiation

Calculations of the absorption of solar radiation by atmospheric gases, and water vapor in particular, are dependent on the quality of databases of spectral line parameters. There has been increasing scrutiny of databases such as HITRAN in recent years, but this has mostly been performed on a band-by-band basis. We report nine high-spectral-resolution (0.03 cm(-1)) measurements of the solar radiation reaching the surface in southern England over the wave number range 2000 to 12,500 cm(-1) (0.8 to 5 mm) that allow a unique assessment of the consistency of the spectral line databases over this entire spectral region. The data are assessed in terms of the modeled water vapor column that is required to bring calculations and observations into agreement; for an entirely consistent database, this water vapor column should be constant with frequency. For the HITRAN01 database, the spread in water vapor column is about 11%, with distinct shifts between different spectral regions. The HITRAN04 database is in significantly better agreement (about 5% spread) in the completely updated 3000 to 8000 cm(-1) spectral region, but inconsistencies between individual spectral regions remain: for example, in the 8000 to 9500 cm(-1) spectral region, the results indicate an 18% (+/- 1%) underestimate in line intensities with respect to the 3000 to 8000 cm(-1) region. These measurements also indicate the impact of isotopic fractionation of water vapor in the 2500 to 2900 cm(-1) range, where HDO lines dominate over the lines of the most abundant isotope of H2O.

Fourier transform infrared spectra of H2CS and D2CS

Infrared spectra of thoformaldehyde, H2CS and D2CS, were observed in the gas phase at a resolution of better than 0.1 cm−1 from 4000 to 400 cm−1 using a Nicolet FTIR system. Vibrational band origins and rotational constants were determined for ν2, ν3, ν4, and ν6 of H2CS and for ν1, ν2, ν3, ν4, and ν6 of D2CS. The ν3, ν4, and ν6 bands of H2CS were analyzed as a set of three Coriolis interacting bands, and three Coriolis constants were determined; similarly the ν4 and ν6 bands of D2CS were analyzed as a pair of interacting bands and one Coriolis constant was determined. A general harmonic force field was determined, without constraints, to fit the vibrational wavenumbers, Coriolis constants, and centrifugal distortion constants. A zero-point (rz) structure was determined from the ground-state rotational constants, and the equilibrium (re) bond lengths were estimated.