The relative influences of acidity and polarity on responsiveness of small organic molecules to analysis with negative ion electrospray ionization mass spectrometry (ESI-MS)

The purpose of the work presented here was to evaluate the influence of solution composition and analyte characteristics on responsiveness to analysis with negative ion electrospray ionization mass spectrometry. The responses of a series of structurally diverse acidic molecules were compared in various solvents. Response was generally observed to be higher in methanol than acetonitrile and response for all analytes was poorer when water was mixed with the organic solvent. A positive correlation between negative ion ESI-MS response and log P was observed when either acetonitrile or methanol was used as the electrospray solvent. This result was expected because analytes with significant nonpolar character should be particularly responsive to ESI-MS analysis due to their higher affinity for electrospray droplet surfaces. It was also predicted that highly acidic analytes would be most responsive to analysis with negative ion ESI-MS due to their tendency to form negative ions. However, for the analytes studied here, acidity was found not to have a consistent influence on ESI-MS response. Many of the highly acidic molecules were quite polar and, consequently, were poorly responsive. Furthermore, the deprotonated molecular ion was detected for a number of molecules with very high pKa values, which would not be expected to form negative ions in the bulk solution. Ultimately, these results indicate that acidity is not a conclusive parameter for prediction of the relative magnitudes of negative ion ESI-MS response among a diverse series of analytes. Analyte polarity does; however, appear to be useful for this purpose.     

Identification of Nitrotyrosine Containing Peptides using Combined Fractional Diagonal Chromatography (COFRADIC) and Off-Line Nano-LC-MALDI

Protein nitration take place on tyrosine residues under oxidative stress conditions and may influence a number of processes including enzyme activity, protein-protein interactions and phospho-tyrosine signalling pathways. Nitrated proteins have been identified in a number of diseases, however, the study of these proteins has been compromised by the lack of good methods for identifying nitrated proteins, their nitration sites and the level of nitration. Here, we present a method for identification of nitrated peptides that allows the site specific assignment of nitration, is easy to use and reproducible, and opens up for the possibility to quantify the level of nitration of specific peptides as function of different oxidative conditions, namely combined fractional diagonal chromatography (COFRADIC) in combination with off-line nano-LC-MALDI. We identify six nitrated peptides from in vitro nitrated bovine serum albumin and propose that automated COFRADIC using nano-LC and off-line MALDI-MS might be a possibility for identification of tyrosine nitrated proteins and the nitration sites in complex samples.     

Homophymine A, an anti-HIV cyclodepsipeptide from the sponge Homophymia sp

A new anti-HIV cyclodepsipeptide, homophymine A, was isolated from a New Caledonian collection of the marine sponge Homophymia sp. The structure of homophymine A was determined by interpretation of spectroscopic data, acid hydrolysis, and LC-MS analysis. Homophymine A contains 11 amino acid residues and an amide-linked 3-hydroxy-2,4,6-trimethyloctanoic acid moiety. Along with four D-, two L-, and one N-methyl amino acids, it also contains four unusual amino acid residues: (2S,3S,4R)-3,4-diMe-Gln, (2R,3R,4S)-4-amino-2,3-dihydroxy-1,7-heptandioic acid, L-ThrOMe, and (2R,3R,4R)-2-amino-3-hydroxy-4,5-dimethylhexanoic acid. In a cell-based XTT assay, homophymine A exhibited cytoprotective activity against HIV-1 infection with a IC50 of 75 nM.     

Relative importance of basicity in the gas phase and in solution for determining selectivity in electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is a critically important technique for the determination of small molecules, but its application for this purpose is complicated by its selectivity. For positive ion ESI-MS analysis of basic analytes, several investigators have pointed to the importance of analyte basicity as a source of selectivity. Currently, however, it is not known whether basicity in the gas phase or in solution is ultimately most important in determining responsiveness. The objective of these studies was to investigate the relative importance of basicity in solution and in the gas phase as factors that predict selectivity in positive ion ESI-MS analysis. ESI-MS response was compared for a diverse series of protonatable analytes in two different solvents, neat methanol and methanol with 0.5% acetic acid. A correlation was observed between analyte pK(b) and electrospray response. However, the response for the analytes with very high pK(b) values was significantly higher than would be expected based on concentration of the protonated form or the analyte in solution, and this higher response did not appear to result from gas-phase proton transfer reactions. Although all of the analytes investigated had higher gas-phase basicities than the solvent, their relative responses were not dictated by gas-phase basicity. Higher response was observed for all of the analytes studied in acidified methanol compared with neat methanol, and this higher response was most pronounced for weakly basic analytes. These findings support the use of analyte pK(b) for rational method development in ESI-MS analysis of small molecules.     

Templated protein assembly on micro-contact-printed surface patterns. Use of the SNAP-tag protein functionality

Micro contact printing (microCP) has been established as a simple technique for high-resolution protein patterning for micro- and nanoarrays. However, as biochemical assays based on immobilized protein arrays progress from immunoassays to more delicate functional assays, the demand for methods of miniaturized, gentle, and oriented immobilization, which are applicable to many different target proteins, becomes larger. In this study, we present a novel microCP templated assembly approach, based on a recombinant SNAP-FLAG-HIS 10 (SFH) immobilization vehicle, which exploits the recently developed SNAP-tag protein. The SNAP-tag is derived from the human DNA repair protein hAGT, which covalently transfers the alkyl group of benzyl guanine (BG) substrates onto itself. We have designed a model SFH cassette carrying three tags (SNAP-tag, FLAG-tag, and HIS-tag), each of which can be used for fluorescence labeling or surface immobilization. When patterns of streptavidin modified with BG-biotin (streptavidin-BG) are stamped onto a surface, the SFH can subsequently assemble on the ligand pattern from solution, functioning as a general immobilization vehicle for high-resolution patterning of any protein expressed in the SFH cassette, in a gentle and oriented manner. Alternatively, the SFH can be site-selectively biotinylated using BG-biotin and, subsequently, assemble on stamped streptavidin. We exploit several ways to biotinylate the SFH protein via the SNAP-tag, promoting its templated assembly on micropatterns of streptavidin in four complementary formats. Quantitative analysis of the obtained patterns, revealed by immunostaining, indicates that all four approaches resulted in proper SFH immobilization and antibody recognition, demonstrating the versatility of the SFH cassette and the potential for high resolution patterning applications. Also, our data confirm that streptavidin can be stamped directly on surfaces, without loss of activity. While three strategies resulted in similar patterning efficiencies, one particular approach--namely templated assembly of SFH directly on streptavidin-BG patterns--resulted in an order of magnitude increase in patterning efficiency.     

Optimizing the stacking moiety and linker of 2'-acylamido caps of DNA duplexes with 3'-terminal adenine residues

Reported here is the synthesis of oligodeoxynucleotides with a 3'-terminal 2'-acylamido-2'-deoxyadenosine residue. The route to these oligonucleotides employs an N,O-Alloc-protected 5'-phosphoramidite of 2'-amino-2'-deoxyadenosine that was prepared in 11 steps from arabinoadenosine. Small combinatorial libraries of oligonucleotides were generated via acylation with a mixture of linker amino acids and subsequent acylation of their amino groups. Mass spectrometrically monitored nuclease selection assays led to oligonucleotides whose 2'-substituent increases the thermal stability of the DNA duplexes. A linker with three methylene groups between a perylene stacking moiety and the amido group gives a UV-melting point increase of up to 27.9 degrees C for the DNA sequence (TGCGCA*)2, where A* denotes the 2'-acylamidoadenosine residue. The same acylamido group improves mismatch discrimination at the terminal position with a melting point depression of >or=7 degrees C for any of the three mismatches in the target sequence of the octamer 5'-AGGTTGAA-3'. These results demonstrate how even a very weakly base-pairing nucleotide at the 3'-terminus of a DNA probe strand can be enforced to engage in strong and highly sequence-selective base-pairing interactions.