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Localized heating of droplets on an electrowetting-on-dielectric (EWOD) chip has been implemented and shown to accelerate trypsin digestion reaction rates, sample drying, and matrix crystallization for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Achieving this involved extending the functionality of previous EWOD droplet-based techniques by developing a multifunctional electrode with closed-loop temperature control, while minimizing overall system complexity and addressing challenges associated with rapid evaporation. For the EWOD chip design, we discuss the performance of multifunctional surface electrodes for actuation, localized Joule heating, and thermistic temperature sensing. Furthermore, a hydrophilic pattern is formed in the multifunctional electrode to control the location of an evaporating droplet on the electrode. To demonstrate the capabilities and limitations of this technique, we performed three experiments and measured the results using MALDI-MS: (i) insulin disulfide reductions in dithiothreitol (DTT) over a range of heater temperatures (22-70[degrees]C) to show how reaction rates can be affected by thermal control, (ii) insulin disulfide reductions at 130[degrees]C in dimethyl sulfoxide (DMSO) to demonstrate a reaction in a high boiling point solvent, and (iii) tryptic digestions of cytochrome c at 22 and 40 [degrees]C to show that heated droplets can yield reasonably higher peptide sequence coverage than unheated droplets. Although they do not decouple the effects of changing temperatures and concentrations, these experiments verified that thermal cycling by EWOD electrodes accelerates reaction rates in liquid droplets in air. 10.1021/ac101833b

Iodination of tyrosine residues in proteins has many uses in chemistry, biology, and medicine. Site specific identification of the sites of iodination is important for many of these uses. Reported herein is a facile method employing photodissociation and mass spectrometry to localize sites of iodination in whole proteins. Absorption of ultraviolet photons by iodotyrosine results in loss of iodine via homolytic bond dissociation. The resulting protein radical fragments in the vicinity of the iodotyrosine upon collisional activation. Analysis of the fragments within the vicinity of each tyrosine residue in the protein enables quantitative evaluation of the likelihood for iodination at each site. The results are compared with both traditional bottom up and top down mass spectrometric methods. Radical directed dissociation yields results in agreement with traditional approaches but requires significantly less effort and is inherently more sensitive. One limitation occurs when multiple tyrosine residues are in close proximity, in which case the extent of iodination at each residue may be difficult to determine. This limitation is frequently problematic for traditional approaches as well. 10.1021/ac100256v

A droplet-based (digital) microfluidics platform has been developed to prepare and purify protein samples for measurement by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Liquid droplets are moved in air by sequentially applying an electric potential to an array of electrodes patterned beneath a hydrophobic dielectric layer. We show that a complete integrated sequence of protein processing steps can be performed on this platform, including disulfide reduction, alkylation, and enzymatic digestion, followed by cocrystallization with a MALDI matrix and analysis of the sample in situ by MALDI-MS. Proteins carbonic anhydrase, cytochrome c, and ubiquitin were used to demonstrate the digestion and postdigestion steps; insulin, serum albumin, and lysezyme were used to illustrate the complete sequence of protein processing steps available with the platform. Several functional improvements in the platform are reported, notably, the incorporation of acetonitrile in the protein droplets to facilitate movement, and patterning the device surfaces to optimize sample crystallization. The method is fast, simple, repeatable, and results in lower reagent consumption and sample loss than conventional techniques for proteomics sample preparation. 10.1021/ac9029373

A number of phosphatidylcholine (PC) cations spanning a mass range of 400-1000 Da are investigated using electrospray ionization mass spectrometry coupled with traveling wave ion mobility spectrometry (TWIMS). A high correlation between mass and mobility is demonstrated with saturated phosphatidylcholine cations in [N.sub.2]. A significant deviation from this mass--mobility correlation line is observed for the unsaturated PC cation. We found that the double bond in the acyl chain causes a 5% reduction in drift time. The drift time is reduced at a rate of ~1% for each additional double bond. Theoretical collision cross sections of PC cations exhibit good agreement with experimentally evaluated values. Collision cross sections are determined using the recently derived relationship between mobility and drift time in TWIMS stacked ring ion guide (SRIG) and compared to estimated collision cross sections using an empiric calibration method. Computational analysis was performed using the modified trajectory (TJ) method with nonspherical [N.sub.2] molecules as the drift gas. The difference between estimated collision cross sections and theoretical collision cross sections of PC cations is related to the sensitivity of the PC cation collision cross sections to the details of the ion--neutral interactions. The origin of the observed correlation and deviation between mass and mobility of PC cations is discussed in terms of the structural rigidity of these molecules using molecular dynamic simulations. 10.1021/ac900672a

Electrospray-assisted laser desorption/ionization (ELDI) is a soft ionization method for mass spectrometry (MS) and combines features of both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization to generate ESI-like multiply charged molecules. The ELDI process is based on merging ESI-generated, charged droplets with particles IN laser desorbed from dried or wet sample deposits. We previously reported that ELDI is amenable for MS-based protein identification of large peptides and small proteins using top-down and bottomup techniques (Peng, I. X.; Shiea, J.; Ogorzalek Lop, R. R.; Lop, J. A. Rapid Commun. Mass Spectrom. 2007, 21, 2541-2546). We have extended our studies by applying collisionally activated dissociation and electron-transfer dissociation MS' to protein analysis and show that ELDI is capable of multistage MS to [MS.sup.4] for top-down characterization of large proteins such as 29 kDa carbonic anhydrase. Multiply charged proteins generated by the ELDI mechanism can be shifted to higher charge by increasing the organic content in the ESI solvent to denature the protein molecules, or by adding m-nitrobenzyl alcohol to the ESI solvent. Furthermore, we introduce 'reactive-ELDI', which supports chemical reactions during the ELDI process. Preliminary data for online disulfide bond reduction using dithiothreitol on oxidized glutathione and insulin show reactive-ELDI to be effective. These data provide evidence that the laser-desorbed particles merge with the ESI-generated charge droplets to effect chemical reactions prior to online MS detection. This capability should allow other chemical and enzymatic reactions to be exploited as online protein characterization tools, as well as extending them to flexible, spatially resolved tissue screening and imaging. Also, these reactive-ELDI disulfide reduction experiments enable direct top-down protein identification for proteomic study, side stepping laborious, time-consuming sample preparation steps such as in-solution reduction and alkylation.

Three MALDI-MS sample/matrix preparation approaches were evaluated for their ability to enhance hydrophobic protein detection from complex mixtures: (1) formic acid-based formulations, (2) perfluorooctanoic acid (PFOA) surfactant addition, and (3) sorbitol addition. While MALDI-MS of Escherichia coli cells desorbed from a standard sinapinic acid matrix displayed 94 [(M + H).sup.+] ions, 119 were observed from a formic acid-based matrix with no more than 10 common to both. Formic acid matrix revealed many lipoproteins and an 8282 m/z ion proposed to be the abundant, water-insoluble ATPase proteolipid. Among the formic acid-based cocktails examined, the slowest rate of serine/threonine formylation was found for 50% [H.sub.2]O/33% 2-propanol/17% formic acid. Faster formylation was observed from cocktails containing more formic acid and from mixtures including C[H.sub.3]CN. Sinapinic, ferulic, DHB, 4-hydroxybenzylidene malononitrile, and 2-mercaptobenzothiazole matrixes performed well in formic acid formulations. Dramatic differences in mixture spectra were also observed from PFOA/ sinapinic acid, at detergent concentrations exceeding the critical micelle concentration, although these matrix cocktails proved difficult to crystallize. E. coli ions observed from these matrix conditions are listed in Tables S-1 and S-3 (Supporting Information). Similar complementarity was observed for M. acetivorans whole-cell mixtures. Including sorbitol in the sinapinic acid matrix was found to promote homogeneous crystallization and to enhance medium and higher m/z ion detection from dilute E. coli cellular mixtures.

The bacterial actin homolog, MreB, forms helical cables within the cell that are required for maintenance of a rod shape. These helical structures are thought to be involved in the spatial organization of cell wall (peptidoglycan) synthesizing complexes of penicillin-binding proteins (PBPs). Here, we examined the role of the MreC cell shape protein in this process in Caulobacter crescentus. Subcellular fractionation experiments showed that MreC is a periplasmic protein and, as assayed by immunofluorescence microscopy, adopted helical or banded patterns along the cell length reminiscent of those formed by MreB and PBP2. The pattern of MreC and PBP2 localization remained when MreB cables were disrupted by treatment with the inhibitor A22. However, long-term absence of MreB led to cell shape changes and an eventual loss of MreC localization, suggesting that an independent structure, perhaps an intact peptidoglycan layer, contributes to the MreC localization pattern. Using affinity chromatography with MreC covalently bound to Sepharose, we isolated several PBPs from cell extracts that eluted from the column as heterogeneous complexes. In this same experiment, using mass spectrometry-based protein identification, we identified several outer membrane proteins, including TonB-dependent receptor transport proteins, that interacted with MreC. Imaging live cells containing fusions of these outer membrane proteins to green fluorescent protein showed that they adopted a subcellular localization pattern that was similar to that of MreC. These results suggest that MreC may function in the spatial organization of PBPs as well as other proteins that lie outside the cytoplasmic membrane. MreB | penicillin binding proteins | peptidoglycan

An in-line sample purification method for MALDI-MS, which relies on the electrowetting-on-dielectric (EWOD)-based technique for digital microfluidics, is reported. In this method, a droplet containing peptides and impurities is moved by EWOD and deposited onto a Teflon-AF surface. A droplet of water is subsequently moved over the spot, where it dissolves and removes the impurities. A droplet containing MALDI matrix is then moved to the spot, which is analyzed by MALDI-MS. This purification method reduces the number of salt adduct peaks caused by low concentrations of impurities (e.g., 20 mM sodium phosphate), and reduces or eliminates the catastrophic effects of high concentrations of impurities (e.g., 8 M urea). The method was used to purify spots made by depositing multiple droplets of contaminated peptides. Spectra from the purified spots showed an increase in the S/N ratio as a function of the number of droplets deposited; when not purified, the S/N ratio remained constant regardless of the number of droplets. Finally, the method was used to purify protein digests for peptide mass fragment (PMF) searches, and was shown to be more efficient than the conventional method of purification with reversed-phase-packed pipet tips. We anticipate this new, in-line sample purification technique for EWOD-MALDI-MS will enable development of integrated high-throughput proteomics analysis methodologies.

Author(s): Sabrina D. Dyall [1, 2]; Weihong Yan [1, 3]; Maria G. Delgadillo-Correa [1]; Adam Lunceford [3]; Joseph A. Loo [4]; Catherine F. Clarke [3]; Patricia J. Johnson (corresponding author) [...]

Loss of function of Bruton's tyrosine kinase (Btk) causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice (xid). By using MS analysis and phosphopeptidespecific antibodies, we identified a tyrosine phosphorylation site (Y617) near the carboxyl terminus of the Btk domain from Btk expressed in 293T as well as DT-40 cells. Y617 is conserved in all Tec family kinases except murine Tec. Replacement of Y617 with a negatively charged glutamic acid (E) suppressed Btk-mediated phospholipase C[gamma]2 activation and calcium response in DT-40 cells, whereas Akt activation was not affected. The Btk Y617E mutant could partially restore conventional B cell development and proliferation in [Btk.sup.-]/[Tec.sup.-] mice but failed to rescue [CD5.sup.+] B-1 cell development and the TI-II immune response to 2,4,6,-trinitrophenyl-Ficoll. These data suggest that Y617 phosphorylation or a negative charge at this site may down-regulate the function of Btk by selectively suppressing the B cell calcium signaling pathway.

A new technique for preparing samples for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported. The technique relies on electrowetting-on-dielectric (EWOD) to move droplets containing proteins or peptides and matrix to specific locations on an array of electrodes for analysis. Standard MALDI-MS reagents, analytes, concentrations, and recipes are demonstrated to be compatible with the technique. Mass spectra are comparable to those collected by conventional methods. Nonspecific adsorption of analytes to device surfaces is demonstrated to be negligible. The results suggest that EWOD may be a useful tool for automating sample preparation for high-throughput proteomics and other applications of MALDI-MS.

Since January 2000, hemoglobin-based oxygen carriers, such as Hemopure, belong to the list of prohibited substances of the International Olympic Committee. Hemopure is based on bovine hemoglobin, which is intra-and intermolecularly cross-linked by glutaraldehyde units causing an average molecular weight of ~250 000. Bovine and human hemoglobins differ by 15% in amino acid sequence; hence, tryptic digestion of these proteins generates species-common and -unique peptides. Those specific fragments originate from the [alpha]- and [beta]-subunits of hemoglobin, such as bovine Hb peptides [[alpha].sub.69-90] (2367.2 Da) or [[beta].sub.40-58] (2089.9 Da). By means of LC-MS/MS, peptides of human and bovine hemoglobin can be separated and identified, enabling the determination of compounds based on Hb of bovine origin and thus the administration of oxygen carriers such as Hemopure. Blank plasma samples were spiked with Hemopure or human or bovine hemoglobin, filtered, enzymatically digested, and analyzed on an Agilent 1100 Series HPLC interfaced to an Applied Biosystems API 2000 triple quadrupole mass spectrometer. In plasma aliquots of 50 [micro]L containing 50 [micro]g of Hemopure (1 mg/mL), peptides of bovine hemoglobin were confirmed, and blank plasma samples as well as 68 specimens of high-performance athletes were tested with the developed procedure.

Frontotemporal lobar degeneration (FTLD) is the third most common neurodegenerative condition after Alzheimer’s and Parkinson’s diseases1. FTLD typically presents in 45 to 64 year olds with behavioural changes or progressive decline of language skills2. The subtype FTLD-TDP is characterized by certain clinical symptoms and pathological neuronal inclusions with TAR DNA-binding protein (TDP-43) immunoreactivity3. Here we extracted amyloid fibrils from brains of four patients representing four of the five FTLD-TDP subclasses, and determined their structures by cryo-electron microscopy. Unexpectedly, all amyloid fibrils examined were composed of a 135-residue carboxy-terminal fragment of transmembrane protein 106B (TMEM106B), a lysosomal membrane protein previously implicated as a genetic risk factor for FTLD-TDP4. In addition to TMEM106B fibrils, we detected abundant non-fibrillar aggregated TDP-43 by immunogold labelling. Our observations confirm that FTLD-TDP is associated with amyloid fibrils, and that the fibrils are formed by TMEM106B rather than TDP-43.Amyloid fibrils extracted from brains of patients with frontotemporal lobar degeneration with TAR DNA-binding-protein immunoreactivity (FTLD-TDP) are made up of transmembrane protein 106B. [ABSTRACT FROM AUTHOR]

Different tauopathies are characterized by the isoform‐specific composition of the aggregates found in the brain and by structurally distinct tau strains. Although tau oligomers have been implicated as important neurotoxic species, little is known about how the primary structures of the six human tau isoforms affect tau oligomerization because the oligomers are metastable and difficult to analyze. To address this knowledge gap, here, we analyzed the initial oligomers formed by the six tau isoforms in the absence of posttranslational modifications or other manipulations using dot blots probed by an oligomer‐specific antibody, native‐PAGE/western blots, photo‐induced cross‐linking of unmodified proteins, mass‐spectrometry, and ion‐mobility spectroscopy. We found that under these conditions, three‐repeat (3R) isoforms are more prone than four‐repeat (4R) isoforms to form oligomers. We also tested whether known inhibitors of tau aggregation affect its oligomerization using three small molecules representing different classes of tau aggregation inhibitors, Methylene Blue (MB), the molecular tweezer CLR01, and the all‐D peptide TLKIVW, for their ability to inhibit or modulate the oligomerization of the six tau isoforms. Unlike their reported inhibitory effect on tau fibrillation, the inhibitors had little or no effect on the initial oligomerization. Our study provides novel insight into the primary–quaternary structure relationship of human tau and suggests that 3R‐tau oligomers may be an important target for future development of compounds targeting pathological tau assemblies. [ABSTRACT FROM AUTHOR]

The enzyme enoyl‐ACP reductase (also called FabI in bacteria) is an essential member of the fatty acid synthase II pathway in plants and bacteria. This enzyme is the target of the antibacterial drug triclosan and has been the subject of extensive studies for the past 20 years. Despite the large number of reports describing the biochemistry of this enzyme, there have been no studies that provided direct observation of the protein and its various ligands. Here we describe the use of native MS to characterize the protein–ligand interactions of FabI with its coenzymes NAD+ and NADH and with the inhibitor triclosan. Measurements of the gas‐phase affinities of the enzyme for these ligands yielded values that are in close agreement with solution‐phase affinity measurements. Additionally, FabI is a homotetramer and we were able to measure the affinity of each subunit for each coenzyme, which revealed that both coenzymes exhibit a positive homotropic allosteric effect. An allosteric effect was also observed in association with the inhibitor triclosan. These observations provide new insights into this well‐studied enzyme and suggest that there may still be gaps in the existing mechanistic models that explain FabI inhibition. [ABSTRACT FROM AUTHOR]