Mass spectrometric methods for identification of protein nitration

Mass spectrometry is a powerful analytical tool that offers some unique benefits when applied to the analysis of 3-nitrotyrosine in proteins. The advantages of mass spectrometric analysis are high mass accuracy, high resolution, high sensitivity, short analysis time and low sample consumption. The application of mass spectrometry as an important tool in biochemical and biomedical science has rapidly increased over the last few years. In 2002, the Nobel Prize for Chemistry was awarded to John Fenn and Koichi Tanaka for the development of “gentle ionization” techniques, electrospray ionization (ESI) [91] and matrix-assisted laser desorption/ ionization (MALDI) [92, 93] mass spectrometry. Both methods facilitate the analysis of biomolecules, such as peptides, proteins and other biochemical compounds, without their destruction and thus opened a way to analyse these molecules [94].

Electrospray ionization (ESI) is a method in which the analyte is sprayed at atmospheric pressure into an interface to the vacuum of the mass spectrometric ion source [91]. The sample solution is sprayed across a high potential difference (1-4kV) from a needle tip into an orifice of the mass analyser. Heat and gas flows may assist in the desolvation of the charged droplets containing the analyte molecular-ions. Finally, ion emission (Taylor-cone-model) leads to the formation of multiply protonated or deprotonated ions (s. Figure 6) [95, 96].

Figure 6: Principle of ionisation source and mechanism of gaseous ion formation in ESI-MS. The sample solution is admitted through a small capillary from which the spray is formed at atmospheric pressure. The charged aerosol is evaporated due to Coulomb explosions to smaller droplets which finally results in desolvated macro-ions.

A major advantage of ESI is that it produces multiply charged ions. Multiple charging allows ions to be analysed based on a mass-to-charge (m/z) ratio, which greatly extends the mass range of the mass analyzer. The number of charges varies, depending on several parameters, including analyte size and structure (shape), solvent, pH, and temperature. For positive ion analysis of peptides and proteins, the charges are normally associated with the most basic amino acids of the molecule and the amino terminus [97, 98]. In fact, the maximum number of charges observed can often be estimated from the primary structure.

Solution flow rates can range from microliters to several millilitres making this ionisation method suitable for interfacing to chromatographic separation methods such as capillary electrophoresis or HPLC. In the last few years several microflow devices have been developed to make possible the protein analysis, available only in very low amounts of sample [99, 100]. Especially nano-electrospray has been shown

Atmospheric pressure Vacuum

Nano-ESI utilizes borosilicate or fused silica glass capillaries that usually have an opening of only 1-10 µm in diameter. These emitters are usually sputter-coated with a conductive material (gold or silver) to allow the high-voltage contact to be made to the tip. In contrast to normal ESI, no pump is used in nano-ESI, and the flow rate is dictated by the potential that is applied to the emitter. Nano-ESI can easily handle submicroliter volumes of samples at flow rates of about 20-50 nl/min. The low flow rates enable enhanced experimental variation which is especially useful for MS / MS experiments and reaction monitoring [101, 102]. For ESI, analysis can be performed on various types of analysers, including (but not limited) quadrupole time-of-flight (QTOF), triple quadrupole, ion trap, or ion cyclotron resonance (ICR).

Matrix-Assisted- Laser-Desorption-Ionisation (MALDI) For laser desorption methods a pulsed laser is used to desorb species from the target surface The incorporation of an analyte into the crystalline structure of small UV-absorbing molecules provided a vehicle for ions to be created from polar or charged biomolecules [103]. The more recent development of MALDI relies on the absorption of laser energy by a solid, microcrystalline matrix compound such as α-cyano-4-hydroxy cinnamic acid or sinapinic acid [92]. To generate gas phase, protonated molecules, a large excess of matrix material is coprecipitated with analyte molecules by pipetting a submicroliter volume of the mixture onto a metal substrate and allowing it to dry. The resulting solid is then irradiated by nanosecond laser pulses, usually from small nitrogen lasers with a wavelength of 337 nm.

Although the details of energy conversion and sample desorption and ionization continues to be studied, a general understanding of the MALDI mechanism is explained below (s. Figure 7). When the laser strikes the matrix crystals, the energy deposition is thought to cause rapid heating of the crystals brought about by matrix molecules emitting absorbed energy in the form of heat. Photoionization of the matrix molecules is also known to occur [92]. The rapid heating causes sublimation of the matrix crystals and expansion of the matrix and analyte into the gas phase. Ions may be formed through gas-phase proton-transfer reactions in the expanding gas phase plume with photo-ionized matrix molecules.

Figure 7: Principle of ionisation/desorption in MALDI-MS. A matrix/analyte- cloud is desorbed from the microcrystalline matrix/sample preparation by a laser pulse. Proton-transfer from matrix ions is thought to be primarily responsible for the subsequent generation of analyte ions.

Normally, low charges are generally produced, even in large biopolymers (e.g. singly and doubly protonated ions), in contrast to the multiply-charged ion structures in ESI-MS [104]. Typically, time-of-flight (TOF) analyzers are employed, but several hybrid systems (QTOF), and high resolution Fourier transform ion cyclotron resonance (FT-ICR) analyzers have been successfully adapted.

The ESI-MS analysis of 3-nitrotyrosine-containing peptides yields unambiguous results, where the introduction of the nitro group increases the molecular weight of the original peptide by +45 atomic mass units (amu). Quantitative analysis of 3-nitrotyrosine-containing peptides can be achieved by ESI-MS analysis using the native reference peptide (NRP) method, i.e., relative to the abundance of unmodified peptides of a given protein of interest [105].

In contrast, under standard UV-MALDI –TOF or FTICR mass spectrometer, using a nitrogen laser (337 nm), a set a photochemical decomposition has been observed, which provide a characteristic pattern for peptides containing 3-nitrotyrosine and therefore may provide problems for the identification of tyrosine nitration in biological materials [106, 107]. The newly introduced infrared-MALDI ionisation, have been developed in our laboratory as powerful approaches for unequivocal and sensitive

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laser pulse matrix/analyte matrix/analyte macro-ion macro-ion hv cloud cluster within cluster

In the last few years the combination of 2D-PAGE, western blotting, and mass spectrometry in a powerful proteomic approach was the more typical strategy to analyze a pattern of nitrated proteins in specific conditions. Aulak and co-workers used 2D gel electrophoresis for the resolution and Western blot detection of 3NT-containing proteins, which were subsequently identified in Database by MALDI-TOF MS [72, 108], but no specific 3-nitrotyrosine residues within these proteins have been reported. A similar methodology was applied by Kanski et al. to analyze the age-dependent accumulation of 3-nitrotyrosine in rat skeletal muscle [109] and heart [74]

and by Turku et al. for the identification of 3-NT-containing proteins in the mitochondrial of diabetic mice [79]. Castegna et al. characterized by a proteomics approach the nitrated proteins in Alzheimer’s disease brain and in the recent paper Sultana et al. investigated the tyrosine nitration associated with proteins in brain of subjects with mild cognitive impairment (MIC) as well (AD); where MCI is considered as a transition phase between control and AD. A characteristic feature of these studies is frequently, that only the protein and not the specific Tyr-nitration structure itself was identified.

Recently, new approach based on a chemical modification of the nitro-tyrosine residues that allows specific labeling of the modified proteins with purification tags followed by selective capturing and enrichment of the labeled proteins, were employed to circumvents some of the limitations associated with the existing immunohistochemical, Western blotting, and chromatography-based methods [110, 111]. New proteomic approaches based on the enrichment strategy with improved derivatisation specificity and high efficiency capture of nitro-tyrosine peptides and the availability of new techniques to specifically map the site of nitration will surely yield useful information in studies of oxidative protein modifications in the near future.

1.4 Problems in using analytical methods for identification of protein