Uncoiling collagen: a multidimensional mass spectrometry study.

Mass spectrometry can be used to determine structural information about ions by activating precursors and analysing the resulting series of fragments. Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) is a technique that correlates the mass-to-charge (m/z) ratio of fragment and precursor ions in a single spectrum. 2D FT-ICR MS records the fragmentation of all ions in a sample without the need for isolation. To analyse specific precursors, horizontal cross-sections of the spectrum (fragment ion scans) are taken, providing an alternative to conventional tandem mass spectrometry (MS/MS) experiments. In this work, 2D FT-ICR MS has been used to study the tryptic digest of type I collagen, a large protein. Fragment ion scans have been extracted from the 2D FT-ICR MS spectrum for precursor m/z ratios: 951.81, 850.41, 634.34, and 659.34, and 2D FT-ICR MS spectra are compared with a set of 1D MS/MS spectra using different fragmentation methods. The results show that two-dimensional mass spectrometry excells at MS/MS of complex mixtures, simplifying spectra by eliminating contaminant peaks, and aiding the identification of species in the sample. Currently, with desktop computers, 2D FT-ICR MS is limited by data processing power, a limitation which should be alleviated using cluster parallel computing. In order to explore 2D FT-ICR MS for collagen, with reasonable computing time, the resolution in the fragment ion dimension is limited to 256k data points (compared to 4M data points in 1D MS/MS spectra), but the vertical precursor ion dimension has 4096 lines, so the total data set is 1G data points (4 Gbytes). The fragment ion coverage obtained with a blind, unoptimized 2D FT-ICR MS experiment was lower than conventional MS/MS, but MS/MS information is obtained for all ions in the sample regardless of selection and isolation. Finally, although all 2D FT-ICR MS peak assignments were made with the aid of 1D FT-ICR MS data, these results demonstrate the promise of 2D FT-ICR MS as a technique for studying complex protein digest mixtures.

A high pressure matrix-assisted laser desorption/ionization Fourier transform mass spectrometry ion source for thermal stabilization of labile biomolecules.

A high pressure matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry (FTMS) ion source was designed and tested. With this design, pressure is pulsed to an estimated 1-10 mbar in the region of the MALDI sample during desorption with the result of significantly decreased fragmentation compared to similar systems operating with pressures of <0.1 mbar. The thermal stabilization of vibrationally excited ions under these conditions is shown with small peptides desorbed from the "hot" matrix alpha-cyano-4-hydroxycinnamic acid, and with the highly labile oxidized beta-chain of insulin. Fragile gangliosides with several sialic acid residues are desorbed under high pressure and remain intact without the typical losses of sialic acid, and a protein standard, ubiquitin (8565.64 Da), is desorbed with minimal dehydration. Under high pressure collisional cooling conditions, non-covalent matrix adduction to the molecular ions becomes prominent, but with the trapped ions in an FT mass spectrometer, the ions can be mildly activated to detach the matrix adducts. The new source, additionally, generates significant levels of the multiply charged ions which are commonly seen in MALDI-TOFMS, but are rarely observed in MALDI-FTMS. This effect is more likely due to the elimination of a mass filtering effect in the previous FTMS ion source than to collisional cooling of the ions.

Matrix-assisted laser desorption/ionization Fourier transform mass spectrometric analysis of oxygenated triglycerides and phosphatidylcholines in egg tempera paint dosimeters used for environmental monitoring of museum display conditions.

Oxidative changes in triacylglycerols and diacylphosphatidylcholines in egg tempera paint strips are used for chemical dosimetry of the quality of the museum environment. High-resolution matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) was used as a rapid method for the determination of the exact elemental composition of the alteration products from diacylphosphatidylcholines and triacylglycerols. Light exposure of the egg tempera paints yields oxygenated diacylphosphatidylcholines and triacylglycerols. In the latter multiple incorporation of oxygen was observed as a recurring mass difference of 15.995, the exact atomic mass of oxygen. Owing to the high resolution of the FTMS data (routinely 20 000 at m/z 1000 in broadband mode), oxidation products with different elemental compositions but identical nominal mass could be distinguished. Products of oxidative cleavage of triacylglycerols were observed in samples exposed for longer times. The relative intensities of the peaks of singly and multiply oxygenated triacylglycerols were used to derive the degree of oxygenation of the egg lipids in the tempera paint dosimeters. The degree of oxygenation was found to be directly related to the light exposure time. Exposure to elevated temperature (60 degrees C) for a period of 21 days did not lead to oxygenation of the triacylglycerols and diacylphosphatidylcholines. Exposure to NO(x) and SO(2) in the dark greatly increased the degree of oxygenation. Addition of lead- or copper-containing pigments to the egg binding medium (and subsequent storage for 6 months in the dark) led to accelerated conversion of egg lipids to oxidised products.

Application of multishot acquisition in Fourier transform mass spectrometry.

A new method of ion injection and trapping is discussed wherein ions are accumulated over several laser shots in the FT-ICR cell prior to detection. This allows accumulation of ion signal without accumulating noise so that the signal/noise ratio is much improved provided that the "space-charge" limit of the total number of ions in the cell is not exceeded. "In-cell" ion accumulation allows selected ion accumulation by simply sweeping unwanted ions out of the cell prior to subsequent ion trapping events and also allows shifted ion accumulations to correct for time-of-flight distortions in the ion abundance distributions.

Internal calibration on adjacent samples (InCAS) with Fourier transform mass spectrometry.

Using matrix-assisted laser desorption/ionization (MAL DI) on a trapped ion mass spectrometer such as a Fourier transform mass spectrometer (FTMS) allows accumulation of ions in the cell from multiple laser shots prior to detection. If ions from separate MALDI samples are accumulated simultaneously in the cell, ions from one sample can be used to calibrate ions from the other sample. Since the ions are detected simultaneously in the cell, this is, in effect, internal calibration, but there are no selective desorption effects in the MALDI source. This method of internal calibration with adjacent samples is demonstrated here on cesium iodide clusters, peptides, oligosaccharides, poly(propylene glycol), and fullerenes and provides typical FTMS internal calibration mass accuracy of < 1 ppm.

Double blind, cluster randomised trial of low dose supplementation with vitamin A or beta carotene on mortality related to pregnancy in Nepal. The NNIPS-2 Study Group.

OBJECTIVE: To assess the impact on mortality related to pregnancy of supplementing women of reproductive age each week with a recommended dietary allowance of vitamin A, either preformed or as beta carotene. DESIGN: Double blind, cluster randomised, placebo controlled field trial. SETTING: Rural southeast central plains of Nepal (Sarlahi district). SUBJECTS: 44 646 married women, of whom 20 119 became pregnant 22 189 times. INTERVENTION: 270 wards randomised to 3 groups of 90 each for women to receive weekly a single oral supplement of placebo, vitamin A (7000 micrograms retinol equivalents) or beta carotene (42 mg, or 7000 micrograms retinol equivalents) for over 31/2 years. MAIN OUTCOME MEASURES: All cause mortality in women during pregnancy up to 12 weeks post partum (pregnancy related mortality) and mortality during pregnancy to 6 weeks postpartum, excluding deaths apparently related to injury (maternal mortality). RESULTS: Mortality related to pregnancy in the placebo, vitamin A, and beta carotene groups was 704, 426, and 361 deaths per 100 000 pregnancies, yielding relative risks (95% confidence intervals) of 0. 60 (0.37 to 0.97) and 0.51 (0.30 to 0.86). This represented reductions of 40% (P<0.04) and 49% (P<0.01) among those who received vitamin A and beta carotene. Combined, vitamin A or beta carotene lowered mortality by 44% (0.56 (0.37 to 0.84), P<0.005) and reduced the maternal mortality ratio from 645 to 385 deaths per 100 000 live births, or by 40% (P<0.02). Differences in cause of death could not be reliably distinguished between supplemented and placebo groups. CONCLUSION: Supplementation of women with either vitamin A or beta carotene at recommended dietary amounts during childbearing years can lower mortality related to pregnancy in rural, undernourished populations of south Asia.

Feasibility of keying data from screen-displayed facsimile images in an ongoing trial: the collaborative ocular melanoma study.

As part of an ongoing clinical trial, we conducted an experiment to assess the feasibility and to determine the reliability of data entry from a computer screen display of images of data collection forms transmitted by facsimile (fax) machines directly into a computer for paper forms designed without consideration of fax or image display requirements. Feasibility was assessed on the basis of accuracy and reliability of data entry and on operator satisfaction. During a 2-week period, half of the forms received at the Collaborative Ocular Melanoma Study (COMS) coordinating center were key-entered twice, using the paper forms as the source (paper source entry). The remaining forms were entered once using paper source and were later reentered using the screen display of images of the faxed forms as the source (image source entry). The latter group of forms, or 50% of all forms received, were entered a third time, using the image source entry. Two data entry operators participated in the experiment. Discrepancy rates between and within data entry operators were calculated for both modes of entry. A total of 50,861 keystrokes (28,095 items) across 1122 records were checked for consistency. The overall discrepancy rate associated with double paper source entry was approximately 21 per 10,000 keystrokes (20 per 10,000 items). Discrepancy rates associated with paper source versus image source entry (53 per 10,000 keystrokes [57 per 10,000 items]) and double image source entry (57 per 10,000 keystrokes [47 per 10,000 items]) were similar in magnitude. Image source entry of forms received by facsimile may provide an acceptable alternative to paper entry in ongoing multicenter clinical trials where the costs of converting existing forms and systems to automated data capture may be unacceptable. This experiment confirmed the feasibility of such an alternative and suggested that improved screen displays and changes in equipment to facilitate entry of data from the screen display may enhance accuracy of entries.

Sequence tag identification of intact proteins by matching tanden mass spectral data against sequence data bases.

Molecular and fragment ion data of intact 8- to 43-kDa proteins from electrospray Fourier-transform tandem mass spectrometry are matched against the corresponding data in sequence data bases. Extending the sequence tag concept of Mann and Wilm for matching peptides, a partial amino acid sequence in the unknown is first identified from the mass differences of a series of fragment ions, and the mass position of this sequence is defined from molecular weight and the fragment ion masses. For three studied proteins, a single sequence tag retrieved only the correct protein from the data base; a fourth protein required the input of two sequence tags. However, three of the data base proteins differed by having an extra methionine or by missing an acetyl or heme substitution. The positions of these modifications in the protein examined were greatly restricted by the mass differences of its molecular and fragment ions versus those of the data base. To characterize the primary structure of an unknown represented in the data base, this method is fast and specific and does not require prior enzymatic or chemical degradation.

Broadband quadrupolar axialization of large multiply charged ions to enhance measurement and minimize conformational restrictions.

For high-resolution Fourier transform mass spectrometry of electrosprayed proteins, the signal-to-noise ratio of measuring nozzle-skimmer fragment ions can be improved substantially by their broadband quadrupolar axialization (QA), even without increasing their concentration in the ion cyclotron resonance cell. Axialization of the product ions makes possible larger, more concentric ion orbits for measurements. QA allowed the identification of new sequence-indicative product ions from a 29 kDa protein. However, QA of large molecular ions gives little increase in signal, consistent with original trapping near the cell axis. By recentering of ions undergoing ion-molecule reactions, these can be carried out at much higher kinetic energy and pressures; for cytochrome c this increases the achievable H-D exchange by 40%, corresponding to exchange at all the active sites of its completely denatured conformer.

Isotopic assignment in large-molecule mass spectra by fragmentation of a selected isotopic peak.

For large ( > 5 kDa) ionic species, Fourier transform ion cyclotron resonance instruments yield by far the highest mass accuracy. However, this can be compromised by misassignment of the isotopic content based on predicted natural abundances of the isotopic peaks. As an alternative method independent of natural abundance variations, high-resolution isolation and dissociation of a single isotopic peak yields a distribution of isotopic peak abundances characteristic of the isotopic content of the precursor peak. Accuracy is enhanced if the precursor peak is abundant and of minimum heavy isotope content, and if the product species is abundant and of intermediate mass. In addition, such spectra of the highest mass products are useful for identifying complementary product pairs, a key step in sequencing proteins and nucleotides.

Gas-phase folding and unfolding of cytochrome c cations.

Water is thought to play a dominant role in protein folding, yet gaseous multiply protonated proteins from which the water has been completely removed show hydrogen/deuterium (H/D) exchange behavior similar to that used to identify conformations in solution. Indicative of the gas-phase accessibility to D2O, multiply-charged (6+ to 17+) cytochrome c cations exchange at six (or more) distinct levels of 64 to 173 out of 198 exchangeable H atoms, with the 132 H level found at charge values 8+ to 17+. Infrared laser heating and fast collisions can apparently induce ions to unfold to exchange at a higher distinct level, while charge-stripping ions to lower charge values yields apparent folding as well as unfolding.

Distinguishing N- and C-terminus ions for mass spectrometry sequencing of proteins without prior degradation.

Identifying complementary pairs of fragment ions, b-type vs y-type, is a key step in mass spectral sequencing of proteins without degradation prior to ionization. Observations that b-type ions tend to be less stable than y-type have been extended to carbonic anhydrase (29 kDa) as a model protein. Infrared multiphoton dissociation (IRMPD) of 24+, 27+, 30+ molecular ions shows y-type ions from complementary pairs require, on average, 27% longer irradiation times for onset of abundance decrease, while in nozzle/skimmer dissociation of all charge states, y-type ions require a 20% higher potential difference. In some cases, b-ion dissociation appears to accompany its formation, as its rate of increase with increasing energy deposition is far less than that of its y-complement.

Complete large-molecule high-resolution mass spectra from 50-femtomole microvolume injection.

For electrospray ionization in Fourier-transform mass spectrometry, direct injection of 5×10(-14) mol (0.5 µL of 100 nM from a microvolume sample valve) of ubiquitin (8565 Da) into the flowing solvent stream yields a spectrum with 85:1 signal-to-noise ratio, 2-ppm mass accuracy, and isotopic resolution. Gated trapping for 100 µs from a 0.15-µL/min injection of 20-µM ubiquitin consumes 5×10(-18) mol, which produces a spectrum with 23:1 signal-to-noise ratio and τ;3×10(5) resolving power.

High-resolution ion isolation with the ion cyclotron resonance capacitively coupled open cell.

For ion cyclotron resonance, a capacitively coupled open cell variant with fourfold radial symmetry was constructed and tested for axial excitation-ejection of large ions at high resolution. With a reverse of frequency sweep direction, this cell gave substantial improvements in signal-to-noise ratio due to linearization of the excitation electric field. Single isotopic peaks of ubiquitin (8.6-kDa) and carbonic anhydrase (29-kDa) molecular ions could be isolated by selective stored waveform inverse Fourier transform excitation, which yielded an order of magnitude higher isolation resolving power than previously achieved at high mass-to-charge ratio values.

Infrared multiphoton dissociation of large multiply charged ions for biomolecule sequencing.

Characterization and verification of the structures of large biomolecules with high-resolution tandem Fourier transform mass spectrometry with electrospray ionization is critically dependent on the technique used to fragment the multiply charged ions produced. Infrared multiphoton dissociation (IRMPD) of ionized proteins as large as carbonic anhydrase (29 kDa) yields fragment information similar to, but with valuable additions to, that of other dissociation techniques. IRMPD yields product ions on-axis, providing efficient dissociation in further stages; MS3 of ubiquitin (8.6 kDa) yields 11 new sequence ions. Optimum irradiation times for protein ion dissociation vary by more than a factor of 6, with times for oligonucleotides far lower, possibly due to photon resonance with a P-O stretching frequency. IRMPD provides far greater selectivity than collisionally activated dissociation and also appears to yield much less mass discrimination and to dissociate much more stable ions. A technique to remove product ions on formation from the laser beam should improve the present efficiencies of 30-80%.