Matrix-assisted Laser Desorption And Ionization Research Articles

Improved peptide mass fingerprinting matches via optimized sample preparation in MALDI mass spectrometry

Peptide mass fingerprinting (PMF) is a powerful technique in which experimentally measured m/z values of peptides resulting from a protein digest form the basis for a characteristic fingerprint of the intact protein. Due to its propensity to generate singly charged ions, along with its relative insensitivity to salts and buffers, matrix-assisted laser desorption and ionization (MALDI)-time-of-flight mass spectrometry (TOFMS) is the MS method of choice for PMF. The qualitative features of the mass spectrum can be selectively tuned by employing different methods to prepare the protein digest and matrix for MALDI-TOFMS. The selective tuning of MALDI mass spectra in order to optimize PMF is addressed here. Bovine serum albumin, carbonic anhydrase, cytochrome c, hemoglobin alpha- and beta-chain, and myoglobin were digested with trypsin and then analyzed by MALDI-TOFMS. 2,5-dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) were prepared using six different sample preparation methods: dried droplet, application of protein digest on MALDI plate followed by addition of matrix, dried droplet with vacuum drying, overlayer, sandwich, and dried droplet with heating. Improved results were obtained for the matrix alpha-cyano-4-hydroxycinnamic acid using a modification of the died droplet method in which the MALDI plate was heated to 80 °C prior to matrix application, which is supported by observations from scanning electron microscopy. Although each protein was found to have a different optimum sample preparation method for PMF, in general higher sequence coverage for PMF was obtained using DHB. The best PMF results were obtained when all of the mass spectral data for a particular protein digest was convolved together.

On‐target derivatization of keratan sulfate oligosaccharides with pyrenebutyric acid hydrazide for MALDI‐TOF/TOF‐MS

In the present work, a rapid and novel method of on-target plate derivatization of keratan sulfate (KS) oligosaccharides for subsequent analysis by matrix-assisted laser desorption and ionization (MALDI) mass spectrometry is described. MALDI-(time-of-flight)-TOF spectra of labeled KS oligosaccharides revealed that significantly improved ionization can be accomplished through derivatization with pyrenebutyric acid hydrazide (PBH), and the most abundant peak in each spectrum corresponds to the singly charged molecular ion [M - H]- or [M + (n - 1)Na - nH]-, where n = the number of sulfates (n = 1, 2, 3...). The high-energy collision-induced dissociation (heCID) spectra of labeled KS oligosaccharides displayed fragments of compounds similar to those observed with laser-induced dissociation (LID) analysis, suggesting that both heCID and LID fragmentations can be used to analyze KS oligosaccharides. Moreover, fragmentation analysis of all labeled KS oligosaccharides was performed by MALDI-TOF/TOF-MS. With LID mode, sodium adducts showed fragmentation of glycosidic linkages with mainly Y/B/C ions, as well as various cross-ring cleavages providing exact information for the positions of sulfate groups along the KS oligosaccharide chains. This one-step on-target derivatization method makes MALDI-TOF/TOF-MS identification of KS fast, simple and highly throughput for trace amounts of biological samples.

Free Radical–Induced Site-Specific Peptide Cleavage in the Gas Phase: Low-Energy Collision-Induced Dissociation in ESI- and MALDI Mass Spectrometry

Protein identification is routinely accomplished by peptide sequencing using mass spectrometry (MS) after enzymatic digestion. Site-specific chemical modification may improve peptide ionization efficiency or sequence coverage in mass spectrometry. We report herein that amino group of lysine residue in peptides can be selectively modified by reaction with a peroxycarbonate and the resulting lysine peroxycarbamates undergo homolytic fragmentation under conditions of low-energy collision-induced dissociation (CID) in electrospray ionization (ESI) and matrix-assisted laser desorption and ionization (MALDI) MS. Selective modification of lysine residue in peptides by our strategy can induce specific peptide cleavage at or near the lysine site. Studies using deuterated analogues of modified lysine indicate that fragmentation of the modified peptides involves apparent free-radical processes that lead to peptide chain fragmentation and side-chain loss. The formation of a-, c-, or z-types of ions in MS is reminiscent of the proposed free-radical mechanisms in low-energy electron capture dissociation (ECD) processes that may have better sequence coverage than that of the conventional CID method. This site-specific cleavage of peptides by free radical- promoted processes is feasible and such strategies may aid the protein sequencing analysis and have potential applications in top-down proteomics.

How to make big molecules fly out of liquid water: applications, features and physics of laser assisted liquid phase dispersion mass spectrometry

Applications, features, and mechanistic details of laser assisted liquid phase dispersion mass spectrometry are highlighted and discussed. It has been used in the past to directly isolate charged molecular aggregates from the liquid phase and to determine their molecular weight employing sensitive time-of-flight mass spectrometry. The liquid matrix in this MALDI (matrix assisted laser desorption and ionization) type approach consists of a 10 microm diameter free liquid filament in vacuum (or a free droplet) which is excited with a focused infrared laser pulse tuned to match the absorption frequency of the OH-stretch vibration of bulk water near 2.8 microm. Due to these features we will refer to the approach as free liquid matrix assisted laser dispersion of ions or ionic aggregates (IR-FL-MALDI), although also LILBID ("laser induced liquid beam (bead) desorption and ionization") has been proposed early as a descriptive acronym for the technique and may be used alternatively. Low-charge-state macromolecular adducts are isolated in the gas phase from solution via a yet poorly characterized mechanism which sensitively depends upon the laser intensity and wavelength, and after the gentle liquid-to-vacuum transfer the aggregates are analyzed via time-of-flight (TOF) mass spectrometry (MS). Possible mechanisms for the isolation and charging of biomolecules directly from liquid solution are discussed in the present contribution. Recent technical advances such as minimizing the sample consumption, strategies for high throughput mass spectrometry, and coupling of liquid beam MS with HPLC will be highlighted as well. An interesting feature of IR-FL-MALDI is what we call the linear response, i.e., a surprising linearity of the gas phase mass signal on the solution concentration over many orders of magnitude for a large number of biomolecular systems as well as ions. Due to these features the approach may be regarded as a true solution probing spectroscopy, which enables elegant biokinetic studies. Several experiments in which time resolved IR-FL-MALDI-MS has recently been employed successfully are given. A particular highlight is the possibility to quantitatively detect oxidation states in solution, which clearly distinguishes the present approach from other established MS source concepts. Due to the good matrix tolerance also proteins in complex mixtures can be monitored quantitatively.

Differential Dimethyl Labeling of N-Termini of Peptides after Guanidination for Proteome Analysis

We describe an enabling technique for proteome analysis based on isotope-differential dimethyl labeling of N-termini of tryptic peptides followed by microbore liquid chromatography (LC) matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS). In this method, lysine side chains are blocked by guanidination to prevent the incorporation of multiple labels, followed by N-terminal labeling via reductive amination using d(0),(12)C-formaldehyde or d(2),(13)C-formaldehyde. Relative quantification of peptide mixtures is achieved by examining the MALDI mass spectra of the peptide pairs labeled with different isotope tags. A nominal mass difference of 6 Da between the peptide pair allows negligible interference between the two isotopic clusters for quantification of peptides of up to 3000 Da. Since only the N-termini of tryptic peptides are differentially labeled and the a(1) ions are also enhanced in the MALDI MS/MS spectra, interpretation of the fragment ion spectra to obtain sequence information is greatly simplified. It is demonstrated that this technique of N-terminal dimethylation (2ME) after lysine guanidination (GA) or 2MEGA offers several desirable features, including simple experimental procedure, stable products, using inexpensive and commercially available reagents, and negligible isotope effect on reversed-phase separation. LC-MALDI MS combined with this 2MEGA labeling technique was successfully used to identify proteins that included polymorphic variants and low abundance proteins in bovine milk. In addition, by analyzing a mixture of two equal amounts of milk whey fraction as a control, it is shown that the measured average ratio for 56 peptide pairs from 14 different proteins is 1.02, which is very close to the theoretical ratio of 1.00. The calculated percentage error is 2.0% and relative standard deviation is 4.6%.

MALDI analysis of presolar nanodiamonds: Mass spectrometric determination of the mass distribution of nanodiamonds from meteorites and a technique to manipulate individual nanodiamonds

This paper describes the use of matrix-assisted laser desorption and ionization (MALDI) to measure the mass distribution of nanodiamonds extracted from meteorites. The techniques used to prepare and mass analyze nanodiamond samples from the Murchison (CM2) and Allende (CV3) meteorites are described. The mass spectra of nanodiamonds (peaking at between 1 × 10^(4)–1.5 × 10^4 Daltons) are compared with size distributions obtained by point-counting transmission electron microscopy (TEM) images obtained elsewhere and reasonable agreement is found. The implications of the ability to produce and mass analyze a beam of nanodiamonds are explored.

High laser-fluence deposition of organic materials in water ice matrices by “MAPLE”

Matrix assisted pulsed laser evaporation (MAPLE) is a deposition technique for organic material. Water ice was used as a matrix for the biotechnologically important guest material, polyethylene glycol (PEG), for concentrations from 0.5 to 4 wt.%. The target was irradiated with 6 ns laser pulses at 355 nm at a fluence of 2.5–12 J/cm 2. Even at this high fluence, Fourier transform infrared spectroscopy (FTIR) indicates a chemical structure of the deposit close to that of the un-irradiated PEG. Matrix assisted laser desorption and ionization (MALDI) and gel permeation chromatography (GPC) show that the mass distribution of the deposited PEG is similar to that of the starting material. Optical pictures of the films show particle structures of PEG of a size up to 5–10 μm. The deposition rate measured with a quartz crystal microbalance is typically of the order of 1 ng/(cm 2 shot).

Serum proteomics profiling—a young technology begins to mature

During the three years since the US National Cancer Institute–Food and Drug Administration (NCI-FDA) proteomics group published their seminal1 (but flawed2–4) study using mass spectrometry to profile the serum proteome of ovarian cancer patients, more than 60 published studies have applied similar technology to a wide range of cancers and other diseases. Although some studies have looked directly at tumor tissue, most have targeted easily accessible fluids such as serum. A few studies used highresolution instruments with a matrix-assisted laser desorption and ionization (MALDI) ion source and a time-of-flight (TOF) ion detector. The majority, however, have relied on lower resolution instruments and surface-enhanced laser desorption and ionization (SELDI), a MALDI variant that uses commercially prepared chromatographic surfaces (ProteinChips; Ciphergen, Fremont, CA, USA) to separate proteins. These studies have a common goal: to identify biomarker patterns in the proteome that can be used for diagnosis, prognosis or monitoring of disease. The preponderance of evidence from these studies seems to suggest that proteomic profiling is able to detect anonymous protein peaks that are expressed differently in cancer patients than in healthy individuals5–7. The studies also provide evidence that we may be able to discover patterns that are useful for prediction of the presence or absence of disease8–11. For serum proteomics to realize its full potential, however, several problems in sensitivity and reproducibility remain unresolved. This article outlines some possible improvements in experimental design and data analysis that might help in this regard.

Mass spectrometry.

AbstractToward the end of the 1980s, two advances in the mass spectrometry (MS) of peptides and proteins were announced independently and, with ongoing developments, are still at the center of current methodologies (1). Both developments were the refinement and application of ionization techniques, namely electrospray and matrix-assisted laser desorption and ionization (MALDI), and both offered increased sensitivity and increased the upper mass limit of peptides or proteins amenable to mass spectrometry. Such was the impact of these techniques that the originators were awarded Nobel Prizes in 2002.KeywordsIonization TechniqueInstrument SettingMass Spectrometry SpectrumHigh Mass AccuracyTriple Quadrupole InstrumentThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Structural characterization of proteins and peptides.

The primary structure of proteins is nowadays determined by DNA sequencing, and a variety of genomes are already known. Nevertheless, protein sequencing/identification is still indispensable to analyze the proteins expressed in a cell, to identify specific proteins, and to determine posttranslational modifications. Proteins of interest are typically available in low microgram amounts or even less. The separation method of choice is gel electrophoresis, followed by blotting to PVDF membrane for N-terminal sequencing or by in-gel digestion to generate peptides that can be separated by HPLC. Structural analysis can be done by Edman degradation or mass spectrometry (MS). Edman degradation is the older method based on successive removal of N-terminal amino acids by chemical methods. Sequencing of a peptide requires many hours, the sensitivity is in the range of 2-5 pmol of a purified peptide. Nevertheless, Edman degradation is still the workhorse in the lab for routine work such as identification of blotted proteins. It is also the method of choice for sequencing unknown proteins/ peptides and modified peptides. MS has routinely been used with peptides in the range of 100 fmol or even less. In contrast to Edman degradation, complex mixtures such as tryptic digests can be analyzed, making HPLC separation of peptides unnecessary. MS is a very fast method that can be automated. It is the method of choice for sensitive analysis and large-scale applications (proteomics). Two different ionization methods are commonly used to generate peptide/protein ions for MS analysis. These are MALDI (matrix assisted laser desorption and ionization) and ESI (electrospray ionization). They can be combined with a variety of mass analyzers (TOF, quadrupole, ion trap). Proteins are either identified by searching databases with the masses of proteolytic peptides (peptide mass fingerprinting) or using fragmentation data (raw MS/MS spectra or sequence tags). This approach requires that the protein is known and listed in the database. De novo sequencing by MS of peptides is possible, but very time consuming and not a routine application, in contrast to Edman degradation. The aim of this chapter is to introduce to basic theory, practical applications and limitations of the various methods, to enable the non-expert scientist to decide which method is best suited for his project and which kind of sample preparation is necessary.

Serum protein MALDI profiling to distinguish upper aerodigestive tract cancer patients from control subjects.

There are no reliable blood markers for the early detection and monitoring of aerodigestive tract tumors. Recent studies have suggested that serum protein patterns may be able to distinguish cancer patients from control subjects. We used matrix-assisted laser desorption and ionization (MALDI) mass spectroscopy to obtain serum protein patterns from patients with head and neck cancer (n = 99) or lung cancer (n = 92) and from control subjects (n = 143) at risk for the development of these cancers. From the mass spectra, we predicted the cancer status of patients using a simple classification procedure based on a t test feature selection and linear discriminant analysis (LDA). We cross-validated the data with 200 random data simulations to establish a range of the LDA tuning parameter, which was used to construct receiver operating characteristic (ROC) curves. Average total protein levels were higher in case patients than in control subjects, although the differences were not statistically significant. Ten individual m/z peaks, from 5 to 111 kd, appeared frequently in head and neck cancer patients but not in control subjects. Using the 45 top predictors, selected by spectral mass and LDA, we observed that ROC curves differed from those expected under the null hypothesis, suggesting that spectral profiles from the sera of patients with head and neck cancer statistically significantly differed from the sera of control subjects. The model developed on head and neck cancer patients could also be used to identify patients with lung cancer. The pattern of protein spectra in total serum reliably distinguished cancer case patients from control subjects. Incorporation of MALDI assays into prospective longitudinal trials to assess the true predictive values of protein spectra in cancer detection is needed.

The Relative Rates of Glutamine and Asparagine Deamidation in Glucagon Fragment 22–29 under Acidic Conditions

Our objective was to compare the relative rates of asparaginyl and glutaminyl deamidation in fragment 22–29 of the polypeptide hormone glucagon in acidic aqueous solutions. Reaction mixtures containing 22–29 (FVQWLMNT) or its degradation products were degraded at 60°C in dilute hydrochloric acid or phosphate buffer in the pH range 1–3. Degradation products were separated by high‐performance liquid chromatography and identified by amino acid sequencing, amino acid analysis, liquid chromatography‐mass spectrometry (LC‐MS), and matrix‐assisted laser desorption and ionization (MALDI). Nine major degradation products were identified, including asparaginyl and glutaminyl deamidated forms, aspartyl peptide cleavage of the asparaginyl deamidated products, and a cyclic imide intermediate. The pH dependences of rate constants for individual pathways were consistent with acid catalysis. Previous investigators have reported a greater susceptibility of asparagine residues to deamidation in neutral and alkaline solutions due to the formation of a more stable five‐membered succinimide intermediate. It has been suggested that asparagine may be more labile under acidic conditions also. We have observed a more facile deamidation for the glutamine residue under the acidic condition studied. It is proposed that the lower reactivity of the asparagine residue may be due to a decreased electrophilicity of its side chain carbonyl carbon imparted by a parallel cleavage pathway at this residue. © 2002 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2332–2345, 2002

Identification of yeast proteins by mass spectrometry

Publisher Summary This chapter discusses the principles and practical protocols involved in the mass spectrometric identification of yeast proteins. Mass spectrometry has improved the toolbox of biochemical strategies by providing rapid and high throughput identification of proteins in yeast. The wide variety of mass spectrometers available commercially can be distinguished by the way ions are produced (ion sources) and the way they are separated (mass analyzers). The most commonly used ion sources for biological applications are matrix-assisted laser desorption and ionization (MALDI) and electrospray (ES) ionization. Both of them are known as “soft ionization methods,” permitting the transfer of the biological compound into the gas phase of the mass analyzer without decomposition. A rapid and sensitive analysis of yeast protein complexes or interacting partners can be achieved by liquid chromatography–mass spectrometry (LC–MS) approaches. The combination of high-resolution mass spectrometry, data-dependent software, and nanoflow liquid chromatography allows the creation of a robust system for the characterization of complex mixtures. The chapter discusses the methods for protein identification.

Soccer-playing metal oxide giant spheres: a first step towards patterning structurally well defined nano-object collectives.

In context with the challenge to assemble giant molecules into patterns with limited size, molybdenum oxide giant spheres (with a molecular mass of about 16 kDa) could be 'kicked out' like soccer balls into the gas phase using matrix assisted laser desorption and ionization (MALDI) and detected by TOF mass spectrometry while cluster collectives ranging from dimers to pentamers were observed.

Vapor deposition of intact polyethylene glycol thin films

Thin films of polyethylene glycol (PEG) of average molecular weight, 1400 amu, were deposited by both matrix-assisted pulsed laser evaporation (MAPLE) and pulsed laser deposition (PLD). The deposition was carried out in vacuum (∼10-6 Torr) with an ArF (λ=193 nm) laser at a fluence between 150 and 300 mJ/cm2. Films were deposited on NaCl plates, Si(111) wafers, and glass slides. The physiochemical properties of the films are compared via Fourier transform infrared spectroscopy (FTIR), electrospray ionization (ESI) mass spectrometry, and matrix-assisted laser desorption and ionization (MALDI) time-of-flight mass spectrometry. The results show that the MAPLE films nearly identically resemble the starting material, whereas the PLD films do not. These results are discussed within the context of biomedical applications such as drug delivery coatings and in vivo applications where there is a need for transfer of polymeric coatings of PEG without significant chemical modification.

Thickness effects of water overlayer on its explosive evaporation at heated metal surfaces

Molecular dynamics (MD) simulations have been employed to investigate the effect of the thickness of a water overlayer on the character of its ejection from a heated Au surface. The simulations are performed for five systems differing in the thickness of the water overlayer which was adsorbed on a metal substrate heated to 1000 K. For each system, an explosive evaporation occurs in the part of the water film adjacent to the metal surface and the upper part of the film is pushed off by the generated force. The average maximum temperature of the water film decreases as the film thickness increases. In contrast, the temperature achieved by the fast cooling due to the explosive evaporation shows an inverse trend. The significance of these model calculations to matrix-assisted laser desorption and ionization (MALDI) mass spectrometry is discussed.

Glycoalkaloid Concentration in Alberta Potatoes Determined by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Matrix-assisted laser desorption and ionization (MALDI) mass spectrometry was used to determine concentrations of individual potato glycoalkaloids in tubers. Samples were extracted with methanol-water and deposited on 2,4,6-trihydroxyacetophenone crystals. Positive ions were analyzed with a MALDI time-of-flight mass spectrometer equipped with a 337 nm laser. Analyte ion intensities relative to an internal standard were used to determine chaconine and solanine concentrations. Calibration curves were prepared by standard additions to potato tuber material. The relative standard deviations (RSDs) of triplicate measurements ranged from 1 to 16%, with an average of 9%. The day-to-day RSD for replicate determinations was 11%. Recoveries of analyst-prepared spikes (50 micrograms/g) averaged 104% for chaconine (RSD, 8%) and 98% for solanine (RSD, 4%). The method limit of detection was estimated to be 2 micrograms/g.

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.

Matrix-assisted laser desorption and ionization in the OH and CO absorption bands of aliphatic and aromatic matrices: dependence on laser wavelength and temporal beam profile

A tunable free-electron laser (FEL) was used to initiate infrared (IR) matrix-assisted laser desorption and ionization (MALDI) of small proteins in aliphatic and aromatic matrices. The laser wavelength was scanned from 2.65 to 4.2 μm and from 5.5 to 6.5 μm, covering the absorption bands of the OH and CO stretching vibrations found in such commonly used IR matrices as succinic, fumaric and nicotinic acids. The temporal profile of the laser pulse was also varied using a broadband electro-optic switch (Pockels cell) to study the effects of fluence and irradiance. Although there are absorption peaks at 3.3 μm for succinic acid and fumaric acid, and at 4.1 μm for nicotinic acid, the lowest threshold-fluence for IR MALDI in this region was around 2.94 μm for all matrices. Moreover, the threshold-fluence increased with increasing absorption up to a value five times that of the 2.94 μm value. This result raises questions about the relative contributions of the different sample constitutents to the absorption and the role of resonant absorption in IR MALDI. The threshold-fluences are typically one order of magnitude higher than those for ultraviolet (UV) MALDI, while extinction coefficients of the IR matrices are 100–1000 times smaller than for UV matrices. Therefore, the absorbed energies per unit volume at the MALDI threshold are 10–100 times smaller than in UV MALDI. All these facts clearly indicate that a different desorption/ionization process must be operative in IR MALDI. Variations in temporal profile of the FEL pulse also revealed that ion desorption depends on laser irradiance rather than laser fluence, a result which cannot be explained simply by energy loss due to heat conduction. Two possible models for IR desorption are suggested based on these observations.

Improved resolution for MALDI-TOF mass spectrometers: a mathematical study

In time-of-flight (TOF) mass spectrometry with matrix-assisted laser desorption and ionization (MALDI), the delayed acceleration of ions (sometimes called “time-lag focusing”, “pulsed ion extraction” or “delayed extraction”) has proven to be a major breakthrough with respect to high mass resolution. This theoretical study presents some focusing modes which improve either the resolution at one mass (spot focusing) or the resolution for all masses simultaneously (wide-range focusing), compared with the normal delayed acceleration mode. The study considers: (1) the case of strong correlation between ion location in space and ion speed at the onset of the accelerating field, observed for low mass MALDI ions, and (2) the case of additional spatial distributions disturbing the space–velocity correlation at higher masses. Improved focusing is achieved by acceleration fields varying in time.