Recent developments in nanoparticle-based MALDI mass spectrometric analysis of phosphoproteomes

Mass spectrometry-based strategies are widely used for mapping of post-translational modifications of phosphoproteins. However, the presence of large amounts of non-phosphopeptides seriously interferes by suppressing the intensities of signals for phosphopeptides in direct MALDI-MS techniques due to the low stoichiometry of protein phosphorylation. Several MALDI-MS approaches are known which use either nanoparticles (NPs) as affinity probes, or NPs as microwave heat absorbers. They assist in the enrichment of trace levels of phosphopeptides from complex protein digests and require minimal sample pretreatment, digestion times, and sample volume. This leads to enhance sensitivity and selectivity in the analysis of the phosphoproteomes. This review (with 89 refs.) summarizes and discusses recent developments in the field, with a particular focus on the potential use of nanomaterials such as metal oxides, metal NPs, NPs-coated target plates, and as core-shell nanocomposites acting as affinity probes and as heat absorbers in MALDI-MS analysis of phosphoproteomes.

Tuning of Ti-doped mesoporous silica for highly efficient enrichment of phosphopeptides in human placenta mitochondria

Extraction of phosphopeptides from rather complex biological samples has been a tough issue for deep and comprehensive investigation into phosphoproteomes. In this paper, we present a series of Ti-doped mesoporous silica (Ti-MPS) materials with tunable composition and controllable morphology for highly efficient enrichment of phosphopeptides. By altering the molar ratio of silicon to titanium (Si/Ti) in the precursor, the external morphology, Ti content, internal long-rang order, and surface area of Ti-MPS were all modulated accordingly with certain regularity. Tryptic digests of standard phosphoprotein α- and β-casein were employed to assess the phosphopeptide enrichment capability of Ti-MPS series. At the Si/Ti molar ratio of 8:1, the optimum enrichment performance with admirable sensitivity and capacity was achieved. The detection limit for β-casein could reach 10 fmol, and 15 phosphopeptides from the digest of α-casein were resolved in the spectrum after enrichment, both superior to the behavior of commercial TiO₂ materials. More significantly, for the digest of human placenta mitochondria, 396 phosphopeptides and 298 phosphoproteins were definitely detected and identified after enrichment with optimized Ti-MPS material, demonstrating its remarkable applicability for untouched phosphoproteomes. In addition, this research also opened up a universal pathway to construct a composition-tunable functional material in pursuit of the maximum performance in applications.

Two-step on-particle ionization/enrichment via a washing- and separation-free approach: multifunctional TiO2 nanoparticles as desalting, accelerating, and affinity probes for microwave-assisted tryptic digestion of phosphoproteins in ESI-MS and MALDI-MS: comparison with microscale TiO2

We introduce a simplified sample preparation method using bare TiO₂ nanoparticles (NPs) to serve as multifunctional nanoprobes (desalting, accelerating, and affinity probes) for effective enrichment of phosphopeptides from microwave-assisted tryptic digestion of phosphoproteins (α-casein, β-casein and milk) in Electrospray Ionization Mass Spectrometry (ESI-MS) and Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS). The results demonstrate that TiO₂ NPs can effectively enrich and accelerate the digestion reactions of phosphoproteins in aqueous solutions and also from complex real samples. After the microwave experiments, we directly injected the resulting solutions into the ESI-MS and MALDI-MS systems for analysis, and excellent sensitivity was achieved without the need for any washing procedure or separation process. The reasons are attributed to the high binding affinity and selectivity of TiO₂ NPs toward phosphopeptides. Thus, phosphopeptides can be adsorbed onto the TiO₂ NP surface. The digested or partially digested phosphoproteins can be concentrated onto the TiO₂ NP surface. This results in the effective or complete digestion of phosphoproteins in a short period of time (45 s). In addition, high sensitivity and sequence coverage of phosphopeptide can be obtained using TiO₂ NPs as microwave absorbers and affinity probes in MALDI-MS and ESI-MS. This is due to the photocatalytic nature of the TiO₂ NPs because the absorption of microwave radiation that can accelerate the activation of trypsin for efficient digestion of phosphoproteins and enhances the ionization of phosphopeptides. The lowest concentrations detected for ESI-MS and MALDI-MS were 0.1 μM and 10 fmol, respectively, for α-casein. Comparing the two-step approach of TiO₂ NPs with microscale TiO₂ particles, the microscale TiO₂ particles shows no effect on the microwave-assisted tryptic digestion of phosphoproteins. The current approach offers multiple advantages, such as great simplicity, high sensitivity and selectivity, straightforward and separation/washing-free technique for phosphorpeptide enrichment analysis.     

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.      

Highly selective and sensitive enrichment of phosphopeptides via NiO nanoparticles using a microwave-assisted centrifugation on-particle ionization/enrichment approach in MALDI-MS

The strategy to concentrate phosphopeptides has become a critical issue for mapping protein phosphorylation sites, which are well known as posttranslational modifications in proteomics. In this study, we propose a simple and highly sensitive method for phosphopeptide enrichment on NiO nanoparticles (NPs) from a trypsin predigested phosphoprotein complex solution in a microwave oven. Furthermore, this technique was combined with centrifugation on-particle ionization/enrichment of phosphopeptides and phosphopeptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Weak magnetism of these NPs and a positive surface charge effect at low pH accomplished rapid and selective phosphopeptide enrichment within 30s. Trypsin-digested products of phosphoproteins such as α-casein and β-casein, human blood serum, nonfat milk, and egg white were also investigated to explore their phosphopeptide enrichment from complex samples by this approach. The results demonstrate that NiO NPs exhibit good affinity to trace the phosphopeptides even in the presence of 30 times higher molar concentration of complex solution of non-phosphopeptide proteolytic predigested bovine serum albumin. The detection limits of NiO NPs for α-casein and β-casein were 2.0?×?10(-9) M, with good signal-to-noise ratio in the mass spectrum. NiO NPs were found to be effective and selective for enrichment of singly and multiply phosphorylated peptides at a trace level in complex samples in a microwave oven. The cost of preparing NiO NPs is low, the NiO NPs are thermally stable, and therefore, they hold great promise for use in phosphopeptide enrichment.     

One-pot synthesis of dopamine dithiocarbamate functionalized gold nanoparticles for quantitative analysis of small molecules and phosphopeptides in SALDI- and MALDI-MS

The sensitivity and efficiency of SALDI-MS or MALDI-MS is mainly dependent on the nature of matrix. A novel approach is proposed for one-pot synthesis of dopamine dithiocarbamate-functionalized gold nanoparticles (DDTC-Au NPs). Their application to quantification of small molecules by surface assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF-MS) and rapid identification of phosphopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is investigated. The synthesized DDTC-Au NPs were characterized by UV-visible and FT-IR spectroscopy, H(1)NMR, SEM and TEM. DDTC-Au NPs offers marked improvement on analyte ionization and effectively suppressed the background noise which leads to clean mass spectra. We also demonstrated the use of DDTC-Au NPs as affinity probes for selective enrichment of phosphopeptides from the solutions of microwave tryptic digested casein proteins. Compared with a conventional matrix, DDTC-Au NPs exhibited a high desorption/ionization efficiency for accurate quantification of small molecules including amino acid (glutathione), drugs (desipramine and enrofloxacin) and peptides (valinomycin and gramicidin D) and successfully utilized as novel affinity probes for straightforward and rapid identification of phosphopeptides from casein proteins (α-, β-casein and nonfat milk), showing a great potentiality to the real-time analysis.     

Fluorometric Beam Profiling of UV MALDI Lasers

The photon distribution (beam profile) of the laser as projected onto the sample is an important variable in matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). Measurement of the beam profile is, therefore, an important factor within MALDI-MS. In this study a simple, low-cost fluorometric laser beam profiling technique is presented and applied in conjunction with MALDI-MS experiments. A comparison of the beam profile information afforded by a commercial system and the fluorometric method is carried out to determine the variation of beam profile for an Nd:YVO₄ laser operated between 1 and 25 kHz. The beam profile information can be used, in conjunction with corresponding ion yields, to inform MALDI-MS experiments. The fluorometric beam profiling technique is used to obtain information about the beam dimensions as incident upon the MALDI-MS sample plate in-source. These values are compared with equivalent information obtained from ablation of thin film α-cyano-4-hydroxycinnamic acid (CHCA). In this study, area estimation by ablation provided a value 1.6 times smaller than that obtained by the fluorometric method, demonstrating the need for caution when measuring beam profile and, therefore, fluence, in MALDI-MS.

Fully automated isotopic dimethyl labeling and phosphopeptide enrichment using a microfluidic HPLC phosphochip

Quantitative detection of phosphorylation levels is challenging and requires an expertise in both stable isotope labeling as well as enrichment of phosphorylated peptides. Recently, a microfluidic device incorporating a nanoliter flow rate reversed phase column as well as a titania (TiO₂) enrichment column was released. This HPLC phosphochip allows excellent recovery and separation of phosphorylated peptides in a robust and reproducible manner with little user intervention. In this work, we have extended the abilities of this chip by defining the conditions required for on-chip stable isotope dimethyl labeling allowing for automated quantitation. The resulting approach will make quantitative phosphoproteomics more accessible.

Microchip-based strategy for enrichment of acetylated proteins

We have developed a simple microchip-based method for the separation and enrichment of acetylated proteins and peptides using a microchip technique. Poly (dimethylsiloxane) (PDMS) microfluidic channels were modified by passing an acidic solution of hydrogen peroxide through them. This resulted in hydrophilic silanol-covered surfaces onto which poly (diallyldimethylammonium chloride) (PDDA) can be coated. Protein A/G beads were then captured by the PDDA layer and antibodies can then be immobilized via the protein A/G. This technique enables efficient capture of antigens due to the optimal spacing and orientation of surface molecules. Two solutions, one containing 72.5 fmol?μL^?1 of acetylated bovine serum albumin (BSA-Ac), the other 72.5 fmol?μL^?1 of tryptic BSA-Ac digest were then enriched. High selectivities were obtained, and a 82.4 % recovery of the acetylated proteins was attained. This on-chip platform was then coupled to MALDI-MS to provide information on the acetylation sites of proteins and peptides. Additional peaks were observed in the mass spectra after enrichment and were assigned to acetylated peptides. This is significant with respect to understanding the mechanism and function of acetylation. In our opinion, this microchip-based technique has a large potential for detecting acetylated proteins and peptides in complex biological mixtures, and in acetylomics in general.

Activated Ion ETD Performed in a Modified Collision Cell on a Hybrid QLT-Oribtrap Mass Spectrometer

We describe the implementation and characterization of activated ion electron transfer dissociation (AI-ETD) on a hybrid QLT-Orbitrap mass spectrometer. AI-ETD was performed using a collision cell that was modified to enable ETD reactions, in addition to normal collisional activation. The instrument manifold was modified to enable irradiation of ions along the axis of this modified cell with IR photons from a CO₂ laser. Laser power settings were optimized for both charge (z) and mass to charge (m/z) and the instrument control firmware was updated to allow for automated adjustments to the level of irradiation. This implementation of AI-ETD yielded 1.6-fold more unique identifications than ETD in an nLC-MS/MS analysis of tryptic yeast peptides. Furthermore, we investigated the application of AI-ETD on large scale analysis of phosphopeptides, where laser power aids ETD, but can produce b- and y-type ions because of the phosphoryl moiety’s high IR adsorption. nLC-MS/MS analysis of phosphopeptides derived from human embryonic stem cells using AI-ETD yielded 2.4-fold more unique identifications than ETD alone, demonstrating a promising advance in ETD sequencing of PTM containing peptides.

Rapid and sensitive detection of bacteria via platinum-labeled antibodies and on-particle ionization and enrichment prior to MALDI-TOF mass spectrometry

We introduce a rapid and sensitive approach to study the interactions of an affinity probe with the bacterial wall. Immunoglobulin was immobilized on platinum nanoparticles, and the resulting probe nanoparticles bind to bacterial walls as confirmed by transmission electron microscopy. A MALDI-MS assay was developed that can detect ~10⁵ cfu mL^?1 of S. marcescens and E. coli. This approach enables simple, rapid and straightforward detection of bacterial proteins, with high resolution and sensitivity, and without the requirement for tedious washing/separation steps.

Sensor for lead(II) ion based on a glassy carbon electrode modified with double-stranded DNA and ferric oxide nanoparticles

Shuttle-like Fe₂O₃ nanoparticles (NPs) were prepared by microwave-assisted synthesis and characterized by scanning electron microscopy and X-ray diffraction. The NPs were immobilized on a glassy carbon electrode and then covered with dsDNA. The resulting electrode gives a pair of well-defined redox peaks for Pb(II) at pH 6.0, with anodic and cathodic peak potentials occurring at ?0.50?V and ?0.75?V (vs. Ag/AgCl), respectively. The amperometric response to Pb(II) is linear in the range from 0.12 to 40?nM, and the detection limit is 0.1?nM at a signal-to-noise ratio of 3. The sensor exhibits high selectivity and reproducibility.

A magnetic copper(II)-imprinted polymer for the selective enrichment of trace copper(II) ions in environmental water

We have developed a convenient, selective and reliable method for the rapid enrichment of trace quantities of Cu(II) by using a magnetic Cu(II) ion-imprinted polymer. This is followed by their determination by FAAS. The imprints were prepared by using (a) Cu(II) ions as the template, (b) 3-aminopropyltriethoxysilane as both the functional monomer and the crosslinking agent, and (c) Fe₃O₄ as the magnetic component. Enrichment is carried out in a single step, and adsorbed copper ions can be separated from the sample solution by applying a strong magnet. The effects of pH, elution condition, amount of imprint, and of potentially interfering ions were evaluated. Under the optimal conditions, the detection limit and enrichment factor are 0.3?μg L^?1 and 100, respectively, and the recovery is >95?%. The procedure was successfully applied in the enrichment and detection of trace copper ions in environmental water.

Iodide-induced adsorption of lead(II) ion on a glassy carbon electrode modified with ferroferric oxide nanoparticles

Spherical Fe₃O₄ nanoparticles (NPs) were prepared by hydrothermal synthesis and characterized by scanning electron microscopy and X-ray diffraction. A glassy carbon electrode was modified with such NPs to result in a sensor for Pb(II) that is based on the strong inducing adsorption ability of iodide. The electrode gives a pair of well-defined redox peaks for Pb(II) in pH 5.0 buffer containing 10 mM concentrations of potassium iodide, with anodic and cathodic peak potentials at ?487 mV and ?622 mV (vs. Ag/AgCl), respectively. The amperometric response to Pb(II) is linear in the range from 0.10 to 44 nM, and the detection limit is 40 pM at an SNR of 3. The sensor exhibits high selectivity and reproducibility.

Rapid analysis of aflatoxin M1 in milk using dispersive liquid–liquid microextraction coupled with ultrahigh pressure liquid chromatography tandem mass spectrometry

A simple, rapid, and sensitive method based on simultaneous protein precipitation and extraction of aflatoxin M₁ (AFM₁) followed by dispersive liquid–liquid microextraction (DLLME) and ultrahigh pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analysis was developed for the determination of AFM₁ in milk samples. In order to precipitate the proteins and extract AFM₁ from milk, a sample pretreatment using acetonitrile and NaCl as the extraction/denaturant solvent and salting-out agent, respectively, was optimised. Subsequently, the acetonitrile (upper) phase, containing AFM₁, was used as the disperser solvent in DLLME, and extractant (chloroform) and water were added in turn to the extract to perform the DLLME process. The main parameters affecting the extraction efficiency of the whole analytical procedure, such as acetonitrile volume, amount of salt, type and volume of extractant and water volume, were carefully optimised by experimental design. Under optimum conditions, the developed method provides an enrichment factor of 33 and detection and quantification limits (0.6 and 2.0 ng kg^?1, respectively) below the maximum levels imposed by current regulations for AFM₁ in milk and infant milk formulae. Recoveries (61.3–75.3 %) and repeatability (RSD?<?10, n?=?3), tested in different types of milk at four AFM₁ levels, met the performance criteria required by EC Regulation No. 401/2006. Moreover, the matrix effect on the signal intensity of the analyte was negligible. The proposed method provides a rapid extraction and an accurate determination of AFM₁ in milk and formula milk using a simple and inexpensive sample preparation procedure.

Ultrasensitive detection of mercury(II) ion using CdTe quantum dots in sol-gel-derived silica spheres coated with calix[6]arene as fluorescent probes

We have developed a simple method for the preparation of highly fluorescent and stable, water-soluble CdTe quantum dots in sol-gel-derived composite silica spheres that were coated with calix[6]arene. The resulting nanoparticles (NP) were characterized in terms of UV, fluorescence and FT-IR spectroscopy and by TEM. The results show that the new NPs display more intense fluorescence intensity and are more stable than its precursors of the type SiO₂/CdTe. In addition, the new NPs exhibit a higher selectivity for the determination of Hg²⁺ than for other metal ions. Under the optimum conditions, the relative fluorescence intensity decreases with the concentration of Hg²⁺ in the range from 2.0 to 14.0?nmol?L^?1 and the detection limit is 1.55?nmol?L^?1. The method is based on the quenching of fluorescence by Hg²⁺ and expected to serve as a practical fluorescence test for rapid detection of Hg²⁺. A mechanism is suggested to explain the inclusion process by a Langmuir binding isotherm, and fluorescence quenching is best described by the Stern-Volmer equation.

Analysis of Biomolecules by Atmospheric Pressure Visible-Wavelength MALDI-Ion Trap-MS in Transmission Geometry

We report the development of a new AP visible-wavelength MALDI-ion trap-MS instrument with significantly improved performance over our previously reported system (Int. J. Mass Spectrom. 315, 66–73 (2012)). A Nd:YAG pulsed laser emitting light at 532 nm was used to desorb and ionize oligosaccharides and peptides in transmission geometry through a glass slide. Limits of detection (LODs) achieved in MS mode correspond to picomole quantities of oligosaccharides and femtomole quantities of peptides. Tandem MS (MS/MS) experiments enabled identification of enzymatically digested proteins and oligosaccharides by comparison of MS/MS spectra with data found in protein and glycan databases. Moreover, the softness of ionization, LODs, and fragmentation spectra of biomolecules by AP visible-wavelength MALDI-MS were compared to those obtained by AP UV MALDI-MS using a Nd:YAG laser emitting light at 355 nm. AP visible-wavelength MALDI appears to be a softer ionization technique then AP UV MALDI for the analysis of sulfated peptides, while visible-wavelength MALDI-MS, MS/MS, and MS/MS/MS spectra of other biomolecules analyzed were mostly similar to those obtained by AP UV MALDI-MS. Therefore, the methodology presented will be useful for MS and MSⁿ analyses of biomolecules at atmospheric pressure. Additionally, the AP visible-wavelength MALDI developed can be readily used for soft ionization of analytes on various mass spectrometers.

Highly sensitive methanol chemical sensor based on undoped silver oxide nanoparticles prepared by a solution method

We have prepared silver oxide nanoparticles (NPs) by a simple solution method using reducing agents in alkaline medium. The resulting NPs were characterized by UV–vis and FT-IR spectroscopy, X-ray powder diffraction, and field-emission scanning electron microscopy. They were deposited on a glassy carbon electrode to give a sensor with a fast response towards methanol in liquid phase. The sensor also displays good sensitivity and long-term stability, and enhanced electrochemical response. The calibration plot is linear (r ²?=?0.8294) over the 0.12?mM to 0.12?M methanol concentration range. The sensitivity is ~2.65?μAcm^?2?mM^?1, and the detection limit is 36.0?μM (at a SNR of 3). We also discuss possible future prospective uses of this metal oxide semiconductor nanomaterial in terms of chemical sensing.

Rapid determination of melamine in milk and milk powder by surface-enhanced Raman spectroscopy and using cyclodextrin-decorated silver nanoparticles

We have synthesized silver nanoparticles (AgNPs) decorated with α-cyclodextrin (CD) by using the traditional silver mirror reaction in the presence of CD. The CD-AgNPs were used as substrate in surface-enhanced Raman spectroscopy (SERS) for determining melamine. The intensity of the Raman band of melamine at 704 cm^?1 was used to determine melamine in milk and milk powder. The use of CD-AgNPs as the SERS substrate rather than classical silver nanoparticles makes the method more sensitive in giving an enhancement by a factor of up to?~?10⁶ in scattering efficiency. The effects of the volume of solutions (of CD-AgNPs, NaCl, NaOH, melamine) and of mixing time were optimized. The standard addition method was employed for quantitative analysis. The correlation coefficient of the calibration plot is 0.9995, and the limit of detection is 3.0 μg L^?1. The method was successfully applied to the determination of melamine in milk and milk powder, with relative standard deviations of <10 % and recoveries between 89 and 104 %.