Streamlined Membrane Proteome Preparation for Shotgun Proteomics Analysis with Triton X-100 Cloud Point Extraction and Nanodiamond Solid Phase Extraction.

While mass spectrometry (MS) plays a key role in proteomics research, characterization of membrane proteins (MP) by MS has been a challenging task because of the presence of a host of interfering chemicals in the hydrophobic protein extraction process, and the low protease digestion efficiency. We report a sample preparation protocol, two-phase separation with Triton X-100, induced by NaCl, with coomassie blue added for visualizing the detergent-rich phase, which streamlines MP preparation for SDS-PAGE analysis of intact MP and shot-gun proteomic analyses. MP solubilized in the detergent-rich milieu were then sequentially extracted and fractionated by surface-oxidized nanodiamond (ND) at three pHs.

Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene.

Acetylene (HCCH) has a long history as a mechanism-based enzyme inhibitor and is considered an active-site probe of the particulate methane monooxygenase (pMMO). Here, we report how HCCH inactivates pMMO in Methylococcus capsulatus (Bath) by using high-resolution mass spectrometry and computational simulation. High-resolution MALDI-TOF MS of intact pMMO complexes has allowed us to confirm that the enzyme oxidizes HCCH to the ketene (C2H2O) intermediate, which then forms an acetylation adduct with the transmembrane PmoC subunit.

Rapid identification of Mycobacterium avium clinical isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Background: Rapid and accurate discrimination of Mycobacterium avium from other mycobacteria is essential for appropriate therapeutic management and timely intervention for infection control. However, routine clinical identification methods for M. avium are both time consuming and labor intensive.

Improved mass spectrometric analysis of membrane proteins based on rapid and versatile sample preparation on nanodiamond particles.

We have developed a novel streamlined sample preparation procedure for mass spectrometric (MS) analysis of membrane proteins using surface-oxidized nanodiamond particles. The platform consists of solid-phase extraction and elution of the membrane proteins on nanodiamonds, concentrating the membrane proteins on the nanodiamonds and separating out detergents, chaotropic agents, and salts, and other impurities that are often present at high concentrations in solubilized membrane preparations. In this manner, membrane-protein extracts are transformed into MS-ready samples in minutes.

Reversible acetylation regulates salt-inducible kinase (SIK2) and its function in autophagy.

Salt-inducible kinase 2 (SIK2) is a serine/threonine protein kinase belonging to the AMP-activated protein kinase (AMPK) family. SIK2 has been shown to function in the insulin-signaling pathway during adipocyte differentiation and to modulate CREB-mediated gene expression in response to hormones and nutrients. However, molecular mechanisms underlying the regulation of SIK2 kinase activity remains largely elusive.

Facile MALDI-MS analysis of neutral glycans in NaOH-doped matrixes: microwave-assisted deglycosylation and one-step purification with diamond nanoparticles.

A streamlined protocol has been developed to accelerate, simplify, and enhance matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) of neutral underivatized glycans released from glycoproteins. It involved microwave-assisted enzymatic digestion and release of glycans, followed by rapid removal of proteins and peptides with carboxylated/oxidized diamond nanoparticles, and finally treating the analytes with NaOH before mixing them with acidic matrix (such as 2,5-dihydroxybenzoic acid) to suppress the formation of both peptide and potassiated oligosaccharide ions in MS analysis. The advantages of this protocol were demonstrated with MALDI-TOF-MS of N-linked glycans released from ovalbumin and ribonuclease B.

Mass production and dynamic imaging of fluorescent nanodiamonds.

Fluorescent nanodiamond is a new nanomaterial that possesses several useful properties, including good biocompatibility, excellent photostability and facile surface functionalizability. Moreover, when excited by a laser, defect centres within the nanodiamond emit photons that are capable of penetrating tissue, making them well suited for biological imaging applications. Here, we show that bright fluorescent nanodiamonds can be produced in large quantities by irradiating synthetic diamond nanocrystallites with helium ions.

Two-photon excited fluorescence of nitrogen-vacancy centers in proton-irradiated type Ib diamond.

Two-photon fluorescence spectroscopy of negatively charged nitrogen-vacancy [(N-V)-] centers in type Ib diamond single crystals have been studied with a picosecond (7.5 ps) mode-locked Nd:YVO(4) laser operating at 1064 nm. The (N-V)- centers were produced by radiation damage of diamond using a 3 MeV proton beam, followed by thermal annealing at 800 degrees C.

Peptide analysis: solid phase extraction-elution on diamond combined with atmospheric pressure matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry.

Electrospray ionization (ESI) has been an indispensable ion generation technique for mass spectrometric analysis of biopolymers such as intact proteins and protein digests operated at atmospheric pressure. Since its advent in 1998, atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) quickly became a popular alternative for the analysis of peptides. Although AP-MALDI sources typically share the same vacuum interface and ion transmission hardware with ESI, it is generally found that ESI is superior in detection sensitivity.

Expression of proteins with dimethylarginines in Escherichia coli for protein-protein interaction studies.

Protein arginine methylation often modulates protein-protein interactions. To isolate a sufficient quantity of proteins enriched in methyl arginine(s) from natural sources for biochemical studies is laborious and difficult. We describe here an expression system that produces recombinant proteins that are enriched in omega-N(G),N(G)-asymmetry dimethylarginines.

Dissociation of heme from gaseous myoglobin ions studied by infrared multiphoton dissociation spectroscopy and Fourier-transform ion cyclotron resonance mass spectrometry.

Detachment of heme prosthetic groups from gaseous myoglobin ions has been studied by collision-induced dissociation and infrared multiphoton dissociation in combination with Fourier-transform ion cyclotron resonance mass spectrometry. Multiply charged holomyoglobin ions (hMbn+) were generated by electrospray ionization and transferred to an ion cyclotron resonance cell, where the ions of interest were isolated and fragmented by either collision with Ar atoms or irradiation with 3 mum photons, producing apomyoglobin ions (aMbn+). Both charged heme loss (with [Fe(III)-heme]+ and aMb(n-1)+ as the products) and neutral heme loss (with [Fe(II)-heme] and aMbn+ as the products) were detected concurrently for hMbn+ produced from a myoglobin solution pretreated with reducing reagents.

Solid-phase extraction and elution on diamond (SPEED): a fast and general platform for proteome analysis with mass spectrometry.

This paper presents a new solid-phase extraction and elution platform based on surface-functionalized diamond nanocrystallites. Compared with conventional methods, the platform facilitates purification and concentration of intact proteins and their enzymatic digests for ensuing sodium dodecyl sulfate-polyacrylamide gel electrophoresis or matrix-assisted laser desorption/ionization mass spectrometry analysis without prior removal of the adsorbent. One-pot work flow involving reduction of disulfide bonds, protection of free cysteine residues, washing off residual chemicals, and proteolytic digestion of adsorbed proteins can be performed directly on the particles.

Polylysine-coated diamond nanocrystals for MALDI-TOF mass analysis of DNA oligonucleotides.

A protocol based on aminated diamond nanocrystals has been developed to isolate, concentrate, purify, and digest DNA oligonucleotides in one microcentrifuge tube for matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. It is shown that use of diamond nanocrystals as a solid-phase extraction support not only permits concentration of oligonucleotides in highly diluted solutions but also facilitates separation of oligonucleotides from proteins in heavily contaminated solutions. Enzymatic digestions can be conducted on particle, and additionally, the digests can be easily recovered from the solution for base sequencing.