Delay Extraction Research Articles

Improvement of peaks identification and dynamic range for bi-polar Single Particle Mass Spectrometer

Delay Extraction (DE) in Single Particle Mass Spectrometry (SPMS) provides substantial resolution enhancement. However, DE has two main drawbacks which are discussed in our article. First, ion peak position in this case becomes very sensitive to initial ion coordinate. Therefore, substantial peak jitter is observed when switching between mass spectra of individual particles. This peak jitter obstructs correct mass spectra accumulation resulting in wide peaks with irregular shapes in the accumulated mass spectrum, which leads to the fact that isotopic pattern identification becomes difficult. In the present article two ways of the peak jitter compensation, Dynamic Calibration and Mass Spectra correction based on Spectra Correlation, are proposed. It was shown that both proposed techniques provide substantial resolution improvement in the summed mass spectrum especially for small peaks with complex isotopic patterns. Second, time delay distorts regular quadratic dependence between time-of-flight and m/z. In our paper we also propose calibration equation with variable exponent for SPMS modified with DE. It is shown that usage of an exponent as a fitting parameter allows to achieve mass accuracy comparable with 4th degree equation proposed earlier.

Observing Proton Transfer Reactions Inside the MALDI Plume: Experimental and Theoretical Insight into MALDI Gas-Phase Reactions.

We evaluated the contribution of gas-phase in-plume proton transfer reactions to the formation of protonated and deprotonated molecules in the MALDI process. A split sample holder was used to separately deposit two different samples, which avoids any mixing during sample preparation. The two samples were brought very close to each other and desorbed/ionized by the same laser pulse. By using a combination of deuterated and non-deuterated matrices, it was possible to observe exclusively in-plume proton transfer processes. The hydrogen/deuterium exchange (HDX) kinetics were evaluated by varying the delayed extraction (DE) time, allowing the desorbed ions and neutrals to interact inside the plume for a variable period of time before being extracted and detected. Quantum mechanical calculations showed that the HDX energy barriers are relatively low for such reactions, corroborating the importance of gas-phase proton transfer in the MALDI plume. The experimental results, supported by theoretical simulations, confirm that the plume is a very reactive environment, where HDX reactions could be observed from 0ns up to 400ns after the laser pulse. These results could be used to evaluate the relevance of previously proposed (and partially conflicting) ionization models for MALDI. Graphical Abstract ᅟ.

Detecting the Site of Phosphorylation in Phosphopeptides without Loss of Phosphate Group Using MALDI TOF Mass Spectrometry

Phosphopeptides with one and four phosphate groups were characterized by MALDI mass spectrometry. The molecular ion of monophosphopeptide could be detected both as positive and negative ions by MALDI TOF with delayed extraction (DE) and in the reflector mode. The tetraphospho peptide could be detected in linear mode. When MS/MS spectra of the monophospho peptides were obtained in a MALDI TOF TOF instrument by CID, b and y ions with the intact phosphate group were observed, in addition the b and y ions without the phosphate group. Our study indicates that it is possible to detect phosphorylated peptides with out the loss of phosphate group by MALDI TOF as well as MALDI TOF TOF instruments with delayed extraction and in the reflector mode.

Theoretical optimization by genetic algorithm of delayed extraction parameters for a matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometer

This paper presents the application of a genetic algorithm (GA) to optimize the operating parameters, namely pulse voltage and extraction delay time, when using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS). Simulations predict the presence of several combinations of these parameters that give a local maximum. The aim is to locate the optimal combination (a global maximum) of pulse voltage and extraction time delay in order to focus the ions of a particular m/z value to achieve the best resolution in a given instrumental geometry. The GA locates the global maximum quickly. The results indicate that it may be possible to achieve very high resolving power by using delayed extraction (DE)-MALDI-TOFMS with parameters obtained from the GA.

Delayed extraction for improved resolution of ion/surface collision products by time-of-flight mass spectrometry

A time-of-flight mass spectrometer is described which contains a collision surface located behind the reflectron. This collision device uses delayed extraction (DE) to improve the instrument resolving power for ion/surface collision products formed by secondary ion mass spectrometry (SIMS) and surface induced dissociation (SID). Comparisons are made between spectra acquired using delayed and continuous extraction. SIMS spectra were acquired by bombarding a perfluoropolyether (PFPE) surface with 1.5 keV ferrocene molecular ion projectiles. Sputtered fluorocarbon ions were detected with resolving powers ( m/Δ m, FWHM) of 150 and 950 using continuous and delayed extraction, respectively. SID spectra were acquired using naphthalene projectile ions which collide upon the PFPE surface at 50 eV. Naphthalene fragments were detected with a resolving power of 130 using continuous extraction and 350 with DE. For both the high- and low-energy experiments, the instrument resolving power showed a strong dependence on extraction delay. Energy resolved SID mass spectra collected using ferrocene molecular ions show that fragmentation of this projectile is similar to that observed while using other instrument geometries.

Analysis of phospho- and glycopolypeptides with infrared matrix-assisted laser desorption and ionization.

The analytical characteristics of infrared (IR) matrix-assisted laser desorption and ionization (MALDI) were investigated for the analysis of phosphopeptides, a phosphopolypeptide, and glycopeptides. Two commercially available instruments, a high-resolution delayed extraction (DE) reflectron time-of-flight (RETOF) mass spectrometer and a high-power pulsed Er:YAG laser, were interfaced to produce a high-resolution MALDI-DE-RETOF instrument that is easy to use and can be switched between UV- and IR-MALDI mode within seconds. In the interface design, particular attention was paid to maintaining the same professional operating environment for the new IR-MALDI mode as exists for the commercial UV-MALDI mode. This instrument configuration facilitates comparative observation and investigation of the relative analytical merits of IR- and UV-MALDI. The results of studies of the tryptic alpha-casein phosphopeptides, RP1 (a Thr45-monophosphorylated congener of the recombinant protein hirudin variant 1), and fetuin Asn81 tryptic glycopeptides are presented. The elimination of labile substituents such as phosphoric acid and sialic acid is suppressed in IR-MALDI-RETOF mass spectrometry, with concomitant higher analyte ion yields. These results reflect the advantages that accrue from deposition of significantly less internal energy in the case of IR-MALDI.

Exploring infrared wavelength matrix-assisted laser desorption/ionization of proteins with delayed-extraction time-of-flight mass spectrometry

We report a study of the application of delayed extraction (DE) to infrared-wavelength matrix-assisted time-of-flight mass spectrometry (IR-MALDI-TOF-MS) of proteins. The shapes of the spectral peaks obtained with DE-IR-MALDI-MS are compared with those obtained from the same samples and matrix using continuous extraction (CE) IR-MALDI-MS. Application of DE results in significant improvements in the peak resolution, revealing spectral features (in proteins with molecular masses <12 kDa) that were not resolved in the corresponding CE-IR-MALDI mass spectra. Particularly significant is a series of peaks on the high mass side of the protonated protein peaks that arise through replacement of protons by adventitious sodium ions in the sample. We deduced that these sodium replacement species are a significant contributer to the broad tails (and resulting peak asymmetries) that are a feature of the DE-IR-MALDI mass spectra of proteins with molecular masses ≥17 kDa. The peak width reduction observed in IR-MALDI by DE suggests that, as in UV-MALDI, the initial velocity distribution for ions produced in the MALDI process contributes to the peak broadness in the CE mass spectra. In a systematic comparison between DE UV-MALDI and DE IR-MALDI, we determined that photochemical matrix adduction is present in UV-MALDI but absent in IR-MALDI. In addition, we find that protein ions produced by IR irradiation are less internally excited (i.e., cooler), exhibiting less fragmentation, more Na + replacement and/or unspecified noncovalent adduction, and more heme adduction with apomyoglobin. Thus, IR-MALDI appears to be a softer means for producing gas-phase protein ions than is UV-MALDI. It will be of considerable practical interest to determine whether large protein ions produced by IR-MALDI are sufficiently cool to survive transport through reflecting TOF mass spectrometers (without loss of small neutral species such as H 2O, NH 3, and CO 2) and the extended time periods required for detection by quadrupole ion trap and Fourier transform ion cyclotron resonance mass analyzers.

Glycoprotein analysis by delayed extraction and post-source decay MALDI-TOF-MS

Matrix-assisted laser desorption-ionization (MALDI)-MS analysis of glycoprotein samples is usually affected by metastable fragmentation, particularly in reflectron instruments. Use of delayed extraction (DE) is shown to minimize the observed metastable fragmentation. In the case of the CHO IL-4, which contains complex-type N-linked glycans, the disialylated component gives the most abundant MALDI signal with a dramatically improved resolution and mass accuracy over the non-DE linear TOF analysis. This increased resolution is advantageous for the 30 kDa Sf9-derived IL-4 receptor, where it is possible to differentiate the individual glycans, but not for higher mass and more heterogeneous glycoproteins. It is also shown that MALDI analysis of proteins with labile functional groups is more superior and reliable with linear DE-TOF systems rather than reflectron TOF analyzers. Further structural information on the sequence and disulfide mapping, as well as glycopeptide identification can be obtained by post-source decay (PSD) analysis of peptide and glycopeptide isolated fractions. The PSD formation of a characteristic triplet of ions separated by 33 Da in mass can be used to identify disulfide-paired peptides, even from complex digest mixtures of proteins.

Delayed extraction time-of-flight MALDI mass spectrometry of proteins above 25,000 Da.

Matrix-assisted laser desorption/ionization (MALDI) with delayed extraction (DE) has been optimized for mass analysis of high-mass proteins and glycoproteins with masses above 25,000 Da. Under optimized experimental conditions, i.e. using a weak extraction field strength and a long delay time, a steep drop in mass resolution above 30,000 Da is no longer observed and an improvement of more than a factor of 10 is obtained compared with the non-DE case, at least up to 66 kDa in a 1.2 m time-of-flight mass analyzer. On this level of resolution the factors limiting further improvements become apparent, i.e. adduct ion formation between matrix and analyte, but also cationization and further non-matrix-related adducts, as well as prompt fragmentation. Moreover, heterogeneity of the sample is often the reason for the detection of broad signals for larger proteins. Within these limitations, DHBs (a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid) gave by far the best results.

On the initial velocity of ions generated by matrix-assisted laser desorption ionization and its effect on the calibration of delayed extraction time-of-flight mass spectra

A novel method was developed to measure the initial velocity of ions generated by matrix-assisted laser desorption ionization (MALDI). It is shown both experimentally and theoretically that with a delayed extraction (DE) technique, the flight time of an ion changes linearly with extraction delay. The initial velocity of the ion, a consequence of the desorption process, can be determined from the slope of this linear curve. Systematic study of the initial velocity was undertaken regarding its dependence on the matrix substance, molecular weight of the analyte, ion polarity, and wavelength of irradiation. It was found that the most important factor was the matrix material. Sinapinic acid and α-cyano-4-hydroxycinnamic acid matrices ejected slower peptide and protein ions than 2,5-dihydroxybenzoic acid or 3-hydroxypicolinic acid: ∼ 300 versus ∼ 550 m/s. Matrix ions themselves exhibited a similar order of initial velocities, but these were 15–40% higher than those of insulin ions. The molecular weight of protein samples (between 5 and 25 ku) was found to have little effect on the initial velocity, but for peptides below 5 ku a gradual transition was noted toward the velocity of the matrix ions. Also decreasing velocity with increasing molecular mass was observed for DNA samples in the 4–14-ku range. In the negative ion mode slightly lower velocities were observed than in the positive ion mode. No difference was found between 337- and 266-nm irradiation. Values of the initial velocities were used to correct systematic errors in the internal calibration observed in mass spectra with delayed extraction. These velocity corrections decrease mass errors substantially in the linear mode, in particular for multicomponent mixtures.

Post-source decay and delayed extraction in matrix-assisted laser desorption/ionization-reflectron time-of-flight mass spectrometry. Are there trade-offs?

By the incorporation of delayed extraction (DE) into matrix-assisted laser desorption/ionization time-of-flight mass spectrometry a dramatic improvement of performance with respect to sensitivity, mass resolution and mass accuracy of precursor ions up to approximately 10 kDa has been achieved. Since DE reduces collisional in-source activation to a large extent, the rate of subsequent metastable decay is considerably reduced. Results are presented which demonstrate that under DE the loss of total post-source decay (PSD) fragment ion yield can be as large as one order of magnitude but that, in terms of sensitivity, part of this loss is balanced by a better S/N ratio which results from a significantly improved mass resolution of the PSD fragment ions (M/delta M up to 1800 compared with M/delta M = 200-500 under prompt extraction). While this compensatory effect is true for the middle to high mass range of PSD fragment ions, it gradually vanishes towards the low mass end of the PSD mass scale where, in the case of linear peptides some important information (immonium ions) is lost. It appears, however, that in the majority of practical PSD work, DE improves the qualty of the PSD spectra and that high energy collisional post-source activation can compensate for the occasional loss of analytical information.

Analysis of Carotenoids and Carotenol Fatty Acid Esters by Matrix-Assisted Laser Desorption Ionization (MALDI) and MALDI–Post-Source-Decay Mass Spectrometry

Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry and MALDI post-source-decay (PSD) fragment ion mass analysis were employed to examine various model carotenoids and some of their fatty acid esters. It was demonstrated that the use of MALDI helps overcome problems resulting from the inherent thermal instability and lack of solubility which render this group of compounds rather difficult for conventional ionization techniques. Detection limits were in the subpicomolar range. Rather abundant metastable fragmentation occurred under conditions of prompt extraction, but was significantly restricted under conditions of delayed extraction (DE). In DE the quasimolecular ion signals (mainly as odd electron radical ions) became the base peak even in spectra of the most delicate fatty acid esters. PSD fragment ion patterns were similar to those recorded under linked B/E scanning in conventional fast atom bombardment–MS and contained information on structural end group substituents such as hydroxyl, epoxide, or carbonyl functions and on the extent of double bond conjugations. The mono- and bis-fatty acid esters fragmented mainly by simple fatty acid cleavage but also furnished some of the end group-specific fragments.