Monitoring the redox cycle of low-molecular peptides using a modified target plate in MALDI-MS

A new method is being proposed for preparing MALDI target plates with a hydrophobic polymer coating and hydrophilic anchors. The particles of the MALDI matrix were pre-mixed with a poly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene] solution prior to their placement on a mass-spectrometric sample support. This technique led to the formation of matrix microspots with a diameter of less than 1 mm inside the polymer. The polymer and matrix concentration as well as the amount of suspension placed on the target plate influenced the size and quality of microspots to a great extent. The sensitivity of the mass-spectrometric analysis was confirmed by obtaining the mass spectra of fmole concentrations of an apomyoglobin tryptic digest. The potential proteomic application of this type of MALDI surface preparation was demonstrated by performing the redox cycle using glutathione and its analogue. All reactions were carried out directly on a MALDI plate, which accommodates low volumes of reagents and prevents sample loss.     

Development of trityl-based photolabile hydroxyl protecting groups

A series of trityl-based photolabile hydroxyl protecting groups have been examined. These PPGs evolve from the traditional acid-labile trityl protecting group with proper electron-donating substituents. Structure-reactivity relationships have been explored. A m-dimethylamino group is crucial to achieve high photochemical deprotection efficiency. The o-hydroxyl group in 8 greatly improves the yield of the photochemical deprotection reaction, compared with the corresponding o-methoxyl-substituted counterpart 7. However, comparison between the photoreactions of 9 and 11 does not show similar structural relevance. The PPG in ether 1 (i.e., DMATr group) is structurally simple and easy to prepare and install. Its deprotection can be successfully carried out with irradiation of sunlight without requirement of photochemical devices.     

Sequential removal of photolabile protecting groups for carbonyls with controlled wavelength

A group of robust and easy-to-make photolabile protecting groups (PPGs) for carbonyl compounds has been developed. Sequential removal of different PPGs is achieved via control of irradiation wavelength.     

Protein micro- and nanopatterning using aminosilanes with protein-resistant photolabile protecting groups

An approach to the integration of nanolithography with synthetic chemical methodology is described, in which near-field optical techniques are used to selectively deprotect films formed by the adsorption of aminosilanes protected by modified 2-nitrophenylethoxycarbonyl (NPEOC) groups. The NPEOC groups are functionalized at the m- or p-position with either a tetraethyleneglycol or a heptaethylene glycol adduct. We describe the synthesis of these bioresistant aminosilanes and the characterization of the resulting photoreactive films. Photodeprotection by exposure to UV light (λ = 325 nm) yielded the amine with high efficiency, at a similar rate for all four adsorbates, and was complete after an exposure of 2.24 J cm(-2). Following photodeprotection, derivatization by trifluoroacetic anhydride was carried out with high efficiency. Micropatterned samples, formed using a mask, were derivatized with aldehyde-functionalized polymer nanoparticles and, following derivatization with biotin, were used to form patterns of avidin-coated polymer particles. Fluorescence microscopy and atomic force microscopy data demonstrated that the intact protecting groups conferred excellent resistance to nonspecific adsorption. Nanometer-scale patterns were created using scanning near-field photolithography and were derivatized with biotin. Subsequent conjugation with avidin-functionalized polymer nanoparticles yielded clear fluorescence images that indicated dense attachment to the nanostructures and excellent protein resistance on the surrounding surface. These simple photocleavable protecting group strategies, combined with the use of near-field exposure, offer excellent prospects for the control of surface reactivity at nanometer resolution in biological systems and offer promise for integrating the top-down and bottom-up molecular fabrication paradigms.     

Highly specific capture and direct MALDI-MS analysis of phosphorylated peptides using novel multifunctional chitosan-GMA-IDA-Fe (III) nanosphere

In this study, we describe a method for highly specific enrichment of phosphopeptides with multifunctional chitosan–glycidyl methacrylate (GMA)–iminodiacetic acid (IDA)–Fe (III) nanospheres for direct analysis by matrix-assisted laser desorption–ionization mass spectrometry (MALDI-MS). This is the first time that chitosan has been used to create nanospheres support material for selective enrichment of phosphopeptides by modification with GMA, derivatization with IDA, and loading with Fe (III) ions. Chitosan-GMA-IDA-Fe (III) nanospheres with a diameter of 20 to 100 nm have multifunctional chemical moieties which confer unique properties, good dispersibility in highly acidic binding buffers, as well as good biocompatibility and chemical stability which improves their specific interaction with phosphopeptides using various types of acid binding buffers. The process of enrichment is very simple, quick, efficient, and specific. Its high specificity and efficiency for purification of phosphopeptides is reflected in the very low and substoichiometric amounts of phosphopeptides which can be detected, in quantities as low as 1:3,000 M ratios. Compared with other state-of the-art technologies such as the use of conventional Fe³⁺-IMAC and TiO₂, these chitosan nanosphere techniques show superior specificity and sensitivity. Moreover, the resultant chitosan-GMA-IDA-Fe³⁺ nanosphere-absorbed phosphopeptides can be either directly analyzed by MALDI-TOF MS analysis or eluted and further analyzed by nano-LC-MS/MS.     

Surface analysis by X-ray photoelectron spectroscopy of sol-gel silica modified with covalently bound peptides

Chemical surface characterization of biologically modified sol-gel derived silica is critical but somewhat limited. This work demonstrates the ability of x-ray photoelectron spectroscopy (XPS) to characterize the surface chemistry of peptide modified sol-gel thin films based on the example of four different free peptide-silanes, denoted RGD, NID, KDI ,and YIG. The N 1s and C 1s peaks were found to be good fingerprints of the peptides, whereas O 1s overlapped with the signal of substrate oxygen and, therefore, the O 1s peak was not informative in the case of the thin films. The C 1s peak was fitted and the contribution of the residual hydrocarbons was sorted out. The curve-fitting procedure of the C 1s peak accounted for the different chemical states of carbon atoms in the peptide structure. The curve-fitting procedure was validated by analyzing free peptides in the powder form and was then applied to the characterization of the peptide-modified thin films. The XPS measured ratio between nitrogen and carbon for the peptide thin film was similar to the corresponding value calculated from the peptide structures. Angle resolved XPS confirmed the surface nature of peptides in modified thin films. The coverage and thickness of the peptides on the thin film surface depended on the peptide sequence. The coverage was in the range of 10% of a monolayer, and the layer thickness varied from 10 to 30 A. We believe that the different thicknesses and surface coverage are due to the local structure of the peptides, with the RGD and NID peptides taking a globule conformation and the YIG and KDI peptides adopting a more linear structure.     

Synthesis and characterization of thiochromone S,S-dioxides as new photolabile protecting groups

3-Arylthiochromone derivatives were synthesized as new photolabile protecting groups, in which the photoreactivity was switchable based on oxidation of the sulfur atom (sulfide and sulfone); the protected substrates , released the corresponding alcohols, amines or carbonxylic acids almost quantitatively under UV-light in neutral condition and the photoproduct showed high fluorescence intensity.     

Multifunctional nanoparticles composite for MALDI-MS: Cd2+-doped carbon nanotubes with CdS nanoparticles as the matrix, preconcentrating and accelerating probes of microwave enzymatic digestion of peptides and proteins for direct MALDI-MS analysis

For the first time, we utilized multifunctional nanoparticles composite (NPs composite) for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis of peptides and proteins. Multiwalled carbon nanotubes doped with Cd(2+) ions and modified with cadmium sulfide NPs were synthesized by a chemical reduction method at room temperature. The multifunctional NPs composite applied for the analysis of peptides and microwave-digested proteins in the atmospheric pressure matrix-assisted laser desorption/ionization ion-trap and MALDI time-of-flight (TOF) mass spectrometry (MS) was successfully demonstrated. The maximum detection sensitivity for peptides in MALDI-MS was achieved by the adsorption of negatively charged peptides onto the surfaces of NP composite through electrostatic interactions. The optimal conditions of peptide mixtures were obtained at 20 min of incubation time using 1 mg of NPs composite when the pH of the sample solution was kept higher than the pI values of peptides. The potentiality of the NP composite in the preconcentration of peptides was compared with that of the individual NP by calculating the preconcentration factors (PF) and found that the NPs composite showed a 4-6 times of PF than the other NPs. In addition, the NPs composite was also applied as heat-absorbing materials for efficient microwave tryptic digestion of cytochrome c and lysozyme from milk protein in MALDI-TOF-MS analysis. We believe that the use of NPs composite technique would be an efficient and powerful preconcentrating tool for MALDI-MS for the study of proteome research.     

Ruthenium(II) bipyridyl complexes as photolabile caging groups for amines

The synthesis and characterization of a series of ruthenium bis(bipyridine) complexes where the inorganic moiety acts as a photolabile protecting group is described. Complexes of the type [Ru(bpy)2L2]+ where bpy = 2,2'-bipyridine and L = butylamine, gamma-aminobutyric acid, tyramine, tryptamine, and serotonin were studied by nuclear magnetic resonance, cyclic voltammetry, and electronic absorption spectroscopy. In all cases, ligands are coordinated by the amine group. The complexes are stable in water for several days and deliver one molecule of ligand upon irradiation with visible light (450 nm). These properties make them suitable for their use as biological caged compounds.     

Elastomeric polyurethanes modified with geminal bisphosphonate groups

Three types of elastomeric segmented polyurethanes represented by a polyether‐urethane, a polyurethane‐urea, and a polycarbonate‐urethane were introduced into a modified low‐temperature variant of base‐induced N‐alkylation of urethane hard segments with an excess of 1,6‐dibromohexane in N,N′‐dimethylacetamide (DMAc), resulting in the modification of polymers with 0.08–0.26 mmol/g of pendant 6‐bromohexyl groups. Either lithium diisopropylamide (LDA) or sodium hydride was used to initiate the reaction, although LDA was found to be more suitable for the bromoalkylation. Selected bromoalkylated polyurethanes of all three types were reacted with thiol‐containing bisphosphonates, to yield the polymers modified with 0.08–0.12 mmol/g of geminal nonesterified covalently attached bisphosphonate groups. Two thiol‐containing geminal bisphosphonates used in the modifications were prepared via reactions of nucleophilic addition to vinylidene‐bisphosphonic acid. All three types of polyurethanes were found equally suitable for the modifications. The bisphosphonate‐modified polyurethanes with nonmetallic cations on the bisphosphonate groups remain soluble in the solvents suitable for the dissolution of nonmodified polymers and can be processed into films by solvent casting. After the exchange of nonmetallic cations to sodium, the polymers become insoluble in any solvent, probably as a result of the intermolecular coordination of bisphosphonate groups with the metal cations. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 39: 105–116, 2001     

o-nitrobenzyl photolabile protecting groups with red-shifted absorption: syntheses and uncaging cross-sections for one- and two-photon excitation

We evaluated the o-nitrobenzyl platform for designing photolabile protecting groups with red-shifted absorption that could be photolyzed upon one- and two-photon excitation. Several synthetic pathways to build different conjugated o-nitrobenzyl backbones, as well as to vary the benzylic position, are reported. Relative to the reference 4,5-dimethoxy-2-nitrobenzyl group, several o-nitrobenzyl derivatives exhibit a large and red-shifted one-photon absorption within the near-UV range. Uncaging after one-photon excitation was studied by measuring UV-visible absorption and steady-state fluorescence emission on model caged ethers and esters. In the whole series investigated, the caged substrates were released cleanly upon photolysis. Quantum yields of uncaging after one-photon absorption lie within the 0.1-1 % range. We observed that these drop as the maximum wavelength absorption of the o-nitrobenzyl protecting group is increased. A new method based on fluorescence correlation spectroscopy (FCS) after two-photon excitation was used to measure the action uncaging cross section for two-photon excitation. The series of o-nitrobenzyl caged fluorescent coumarins investigated exhibit values within the 0.1-0.01 Goeppert-Mayer (GM) range. Such results are in line with the low quantum yields of uncaging associated with cross-sections of 1-50 GM for two-photon absorption. Although the cross-sections for one- and two-photon absorption of o-nitrobenzyl photolabile protecting groups can be readily improved, we emphasize the difficulty in enlarging the corresponding action uncaging cross-sections in view of the observed trend of their quantum yield of uncaging.     

Direct MALDI-MS analysis of cardiolipin from rat organs sections

Cardiolipins (CL) are mitochondria specific lipids. They play a critical role in ATP synthesis mediated by oxidative phosphorylation. Abnormal CL distribution is associated with several disease states. MALDI-MS and MALDI-MS/MS were used to demonstrate in situ analysis and characterization of CL from tissue sections of organs containing high concentrations of mitochondria. Once the experimental parameters were established, a survey of CL distribution in heart, liver, kidney, leg muscle, and testis was undertaken. The major CL specie in the heart muscle, leg muscle, liver, and kidney is the (18:2)(4) CL, while liver and kidney also contain a minor specie, (18:2)(3)/(18:1) CL. The major CL specie in testis is the (16:0)(4) CL. The CL species distribution in various organs appeared to be in agreement with prior reports. Overall, proper matrix selection, tissue section handling, instrument tuning, and the inclusion of cesium ion in matrix ensured successful in situ MALDI-MS and MALDI-MS/MS analysis of CL. Upon modification and standardization, this method could be streamlined for rapid pathological diagnosis with short turnaround time in clinical settings.     

Diagonal chromatographic selection of cysteinyl peptides modified with benzoquinones

The derivatization of cysteine-containing peptides with benzoquinone compounds is rapid, quantitative and specific in acidic media. The conversion of cysteines into hydrophobic benzoquinone-adducted residues in peptides is used here to alter the chromatographic properties of cysteinyl peptides during liquid chromatography separation. The benzoquinone derivatization is shown to allow the accurate selection of cysteine-containing peptides of bovine serum albumin tryptic digest by diagonal reversed-phase chromatography, which consists of one primary and a series of secondary identical liquid chromatographic separations, before and after a cysteinyl-targeted modification of the peptides by benzoquinone compounds. Figure Diagonal chromatographic selection of cysteinyl peptides modified with benzoquinones Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Multifunctional ZrO2 nanoparticles and ZrO2-SiO2 nanorods for improved MALDI-MS analysis of cyclodextrins, peptides, and phosphoproteins

Multifunctional ZrO₂ nanoparticles (NPs) and ZrO₂-SiO₂ nanorods (NRs) have been successfully applied as the matrices for cyclodextrins and as affinity probes for enrichment of peptides (leucine-enkephalin, methionine-enkephalin and thiopeptide), phosphopeptides (from tryptic digestion products of β-casein) and phosphoproteins from complex samples (urine and milk) in atmospheric pressure matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and MALDI time-of-flight (TOF) MS. The results show that the ZrO₂ NPs and ZrO₂-SiO₂ NRs can interact with target molecules (cyclodextrins, peptides, and proteins), and the signal intensities of the analytes were significantly improved in MALDI-MS. The maximum signal intensities of the peptides were obtained at pH 4.5 using ZrO₂ NPs and ZrO₂-SiO₂ NRs as affinity probes. The limits of detection of the peptides were found to be 75-105 fmol for atmospheric pressure MALDI-MS and those of the cyclodextrins and β-casein were found to be 7.5-20 and 115-125 fmol, respectively, for MALDI-TOF-MS. In addition, these nanomaterials can be applied as the matrices for the analysis of cyclodextrins in urine samples by MALDI-TOF-MS. ZrO₂ NPs and ZrO₂-SiO₂ NRs efficiently served as electrostatic probes for peptide mixtures and milk proteins because 2–11 times signal enhancement can be achieved compared with use of conventional organic matrices. Moreover, we have successfully demonstrated that the ZrO₂ NPs can be effectively applied for enrichment of phosphopeptides from tryptic digestion of β-casein. Comparing ZrO₂ NPs with ZrO₂-SiO₂ NRs, we found that ZrO₂ NPs exhibited better affinity towards phosphopeptides than ZrO₂-SiO₂ NRs. Furthermore, the ZrO₂ and ZrO₂-SiO₂ nanomaterials could be used to concentrate trace amounts of peptides/proteins from aqueous solutions without tedious washing procedures. This approach is a simple, straightforward, separation-and washing-free approach for MALDI-MS analysis of cyclodextrins, peptides, proteins, and tryptic digestion products of phosphoproteins.      

Toward the development of new photolabile protecting groups that can rapidly release bioactive compounds upon photolysis with visible light

The synthesis and characterization of a new photolabile protecting group (caging group) for carboxylic acids, the 2-(dimethylamino)-5-nitrophenyl (DANP) group, is described. This compound has a major absorption band in the visible wavelength region with a maximum near 400 nm (epsilon400 = 9077 M(-1) cm(-1) at pH 7.4 and 21 degrees C). The caging group is attached through an ester linkage to the carboxyl functionality of beta-alanine, which activates the inhibitory glycine receptor in the mammalian central nervous system. Such caged compounds play an important role in transient kinetic investigations of fast cellular processes. Upon photolysis of DANP-caged beta-alanine, the caging group is released within 5 micros. Quantum yields of 0.03 and 0.002 were obtained in the UV region (308 and 360 nm) and the visible region (450 nm), respectively. Laser-pulse photolysis experiments, using 337 or 360 nm light, were performed with the caged compound equilibrated with HEK 293 cells transiently transfected with cDNA encoding the alpha1 homomeric, wild-type glycine receptor. The experiments demonstrated that neither DANP-caged beta-alanine nor its byproducts inhibit or activate the glycine receptors on the cell surface. Under physiological conditions, the DANP-caged beta-alanine is water-soluble and stable and can be used for transient kinetic measurements.     

Oxidation with a photolabile carbonyl protecting group

A novel oxidation approach utilizing a robust photolabile carbonyl protecting group reagent (1) as the oxidizing reagent has been developed. Different from existing methods, this approach oxidizes primary alcohols to the photosensitive acetals (e.g., 3), providing another unique approach to the protected aldehydes. Thus, for the first time, oxidation and protection are achieved in one reaction. Secondary alcohols are oxidized to the corresponding ketones. Moreover, the photolabile protecting group (PPG) also oxidizes ethers and esters. The oxidation is presumably via hydride abstraction by the tritylium ion generated from 1 under acidic conditions. However, the mechanisms for primary alcohols and secondary alcohols are slightly different.     

Robust ion-permselective multilayer films prepared by photolysis of polyelectrolyte multilayers containing photo-cross-linkable and photolabile groups

The azobenzene-containing polyanion PAC-azoBNS was alternately assembled with the polycation diazoresin (DAR) to construct photo-cross-linkable multilayer films of PAC-azoBNS/DAR that contain photolabile groups of azobenzene. Upon mild UV irradiation, the interaction between PAC-azoBNS/DAR multilayers was converted from electrostatic interaction to covalent bonds. Because of the free carboxylic acid groups presented in the film, the photo-cross-linked multilayer film favors the selective permeation of positively charged species. After photolysis of the photo-cross-linked PAC-azoBNS/DAR films with intense UV irradiation, azobenzene groups decompose to produce imine groups, and a photo-cross-linked robust film containing free carboxylic acid and imine groups was fabricated. The resultant film allows the permeation of negatively charged species and meanwhile shows a pH-switchable permselectivity for positively charged species. Because of the covalently cross-linking structure, the photolyzed cross-linked PAC-azoBNS/DAR film shows high reversible switching behavior and has high stability in solution with high ionic strength.