Hybridizing Ultraviolet Photodissociation with Electron Transfer Dissociation for Intact Protein Characterization

We report a hybrid fragmentation method involving electron transfer dissociation (ETD) combined with ultraviolet photodissociation (UVPD) at 193 nm for analysis of intact proteins in an Orbitrap mass spectrometer. Integrating the two fragmentation methods resulted in an increase in the number of identified <i>c</i>- and <i>z</i>-type ions observed when compared to UVPD or ETD alone, as well as generating a more balanced distribution of <i>a</i>/<i>x</i>,<i> b</i>/<i>y</i>, and <i>c</i>/<i>z</i> ion types. Additionally, the method was shown to decrease spectral congestion via fragmentation of multiple (charge-reduced) precursors. This hybrid activation method was facilitated by performing both ETD and UVPD within the higher energy collisional dissociation (HCD) cell of the Orbitrap mass spectrometer, which afforded an increase in the total number of fragment ions in comparison to the analogous MS³ format in which ETD and UVPD were undertaken in separate segments of the mass spectrometer. The feasibility of the hybrid method for characterization of proteins on a liquid chromatography timescale characterization was demonstrated for intact ribosomal proteins

Improvement of Shotgun Proteomics in the Negative Mode by Carbamylation of Peptides and Ultraviolet Photodissociation Mass Spectrometry

Although acidic peptides compose a substantial portion of many proteomes, their less efficient ionization during positive polarity electrospray ionization (ESI) impedes their detection in bottom-up mass spectrometry workflows. We have implemented a derivatization strategy based on carbamylation which converts basic amine sites (Lys, N-termini) to less basic amides for enhanced analysis in the negative mode. Ultraviolet photodissociation (UVPD) is used to analyze the resulting peptide anions, as demonstrated for tryptic peptides from bovine serum albumin and <i>Halobacterium salinarum</i> in a high throughput liquid chromatography/tandem mass spectrometry (LC/MS/MS) mode. LC/UVPD-MS of a carbamylated <i>H. salinarum</i> digest resulted in 45% more identified peptides and 25% more proteins compared to the unmodified digest analyzed in the negative mode

De Novo Sequencing of Peptides Using Selective 351 nm Ultraviolet Photodissociation Mass Spectrometry

Although in silico database search methods remain more popular for shotgun proteomics methods, de novo sequencing offers the ability to identify peptides derived from proteins lacking sequenced genomes and ones with subtle splice variants or truncations. Ultraviolet photodissociation (UVPD) of peptides derivatized by selective attachment of a chromophore at the N-terminus generates a characteristic series of y ions. The UVPD spectra of the chromophore-labeled peptides are simplified and thus amenable to de novo sequencing. This method resulted in an observed sequence coverage of 79% for cytochrome C (eight peptides), 47% for β-lactoglobulin (five peptides), 25% for carbonic anhydrase (six peptides), and 51% for bovine serum albumin (33 peptides). This strategy also allowed differentiation of proteins with high sequence homology as evidenced by de novo sequencing of two variants of green fluorescent protein

Top-Down 193-nm Ultraviolet Photodissociation Mass Spectrometry for Simultaneous Determination of Polyubiquitin Chain Length and Topology

Protein ubiquitin modifications present a vexing analytical challenge, because of the dynamic changes in the site of modification on the substrate, the number of ubiquitin moieties attached, and the diversity of linkage patterns in which they are attached. Presented here is a method to confidently assign size and linkage type of polyubiquitin modifications. The method combines intact mass measurement to determine the number of ubiquitin moieties in the chain with backbone fragmentation by 193-nm ultraviolet photodissociation (UVPD) to determine the linkage pattern. UVPD fragmentation of proteins leads to reproducible backbone cleavage at almost every inter-residue position, and in polyubiquitin chains, the N-terminally derived fragments from each constituent monomer are identical, up to the site of conjugation. The N-terminal ubiquitin fragment ions are superimposed to create a diagnostic pattern that allows easy recognition of the dominant chain linkages. The method is demonstrated by achieving almost-complete fragmentation of monoubiquitin and then, subsequently, fragmentation of dimeric, tetrameric, and longer Lys48- and Lys63-linked ubiquitin chains. The utility of the method for the analysis of mixed linkage chains is confirmed for mixtures of Lys48 and Lys63 tetramers with known relative concentrations and for an <i>in vitro</i>-formulated ubiquitin chain attached to a substrate protein

Top-Down 193-nm Ultraviolet Photodissociation Mass Spectrometry for Simultaneous Determination of Polyubiquitin Chain Length and Topology

Protein ubiquitin modifications present a vexing analytical challenge, because of the dynamic changes in the site of modification on the substrate, the number of ubiquitin moieties attached, and the diversity of linkage patterns in which they are attached. Presented here is a method to confidently assign size and linkage type of polyubiquitin modifications. The method combines intact mass measurement to determine the number of ubiquitin moieties in the chain with backbone fragmentation by 193-nm ultraviolet photodissociation (UVPD) to determine the linkage pattern. UVPD fragmentation of proteins leads to reproducible backbone cleavage at almost every inter-residue position, and in polyubiquitin chains, the N-terminally derived fragments from each constituent monomer are identical, up to the site of conjugation. The N-terminal ubiquitin fragment ions are superimposed to create a diagnostic pattern that allows easy recognition of the dominant chain linkages. The method is demonstrated by achieving almost-complete fragmentation of monoubiquitin and then, subsequently, fragmentation of dimeric, tetrameric, and longer Lys48- and Lys63-linked ubiquitin chains. The utility of the method for the analysis of mixed linkage chains is confirmed for mixtures of Lys48 and Lys63 tetramers with known relative concentrations and for an <i>in vitro</i>-formulated ubiquitin chain attached to a substrate protein

Synthesis and Self-Assembly Processes of Monofunctionalized Cucurbit[7]uril

We present a building-block approach toward functionalized CB[7] derivatives by the condensation of methylene-bridged glycoluril hexamer <b>1</b> and glycoluril bis­(cyclic ethers) <b>2</b> and <b>12</b>. The CB[7] derivatives Me₂CB­[7] and CyCB[7] are highly soluble in water (264 mM and 181 mM, respectively). As a result of the high intrinsic solubility of Me₂CB­[7], it is able to solubilize the insoluble benzimidazole drug albendazole. The reaction of hexamer <b>1</b> with glycoluril derivative <b>12</b>, which bears a primary alkyl chloride group, gives CB[7] derivative <b>18</b> in 16% isolated yield. Compound <b>18</b> reacts with NaN₃ to yield azide-substituted CB[7] <b>19</b> in 81% yield, which subsequently undergoes click reaction with propargylammonium chloride (<b>21</b>) to yield CB[7] derivative <b>20</b> in 95% yield, which bears a covalently attached triazolyl ammonium group along its equator. The results of NMR spectroscopy (¹H, variable-temperature, and DOSY) and electrospray mass spectrometry establish that <b>20</b> undergoes self-assembly to form a cyclic tetrameric assembly (<b>20</b>₄) in aqueous solution. CB[7] derivatives bearing reactive functional groups (e.g., N₃, Cl) are now available for incorporation into more complex functional systems

Synthesis and Self-Assembly Processes of Monofunctionalized Cucurbit[7]uril

We present a building-block approach toward functionalized CB[7] derivatives by the condensation of methylene-bridged glycoluril hexamer <b>1</b> and glycoluril bis­(cyclic ethers) <b>2</b> and <b>12</b>. The CB[7] derivatives Me₂CB­[7] and CyCB[7] are highly soluble in water (264 mM and 181 mM, respectively). As a result of the high intrinsic solubility of Me₂CB­[7], it is able to solubilize the insoluble benzimidazole drug albendazole. The reaction of hexamer <b>1</b> with glycoluril derivative <b>12</b>, which bears a primary alkyl chloride group, gives CB[7] derivative <b>18</b> in 16% isolated yield. Compound <b>18</b> reacts with NaN₃ to yield azide-substituted CB[7] <b>19</b> in 81% yield, which subsequently undergoes click reaction with propargylammonium chloride (<b>21</b>) to yield CB[7] derivative <b>20</b> in 95% yield, which bears a covalently attached triazolyl ammonium group along its equator. The results of NMR spectroscopy (¹H, variable-temperature, and DOSY) and electrospray mass spectrometry establish that <b>20</b> undergoes self-assembly to form a cyclic tetrameric assembly (<b>20</b>₄) in aqueous solution. CB[7] derivatives bearing reactive functional groups (e.g., N₃, Cl) are now available for incorporation into more complex functional systems

Enrichment of Plasma Membrane Proteins Using Nanoparticle Pellicles: Comparison between Silica and Higher Density Nanoparticles

Proteomic and other characterization of plasma membrane proteins is made difficult by their low abundance, hydrophobicity, frequent carboxylation, and dynamic population. We and others have proposed that underrepresentation in LC-MS/MS analysis can be partially compensated by enriching the plasma membrane and its proteins using cationic nanoparticle pellicles. The nanoparticles increase the density of plasma membrane sheets and thus enhance separation by centrifugation from other lysed cellular components. Herein, we test the hypothesis that the use of nanoparticles with increased densities can provide enhanced enrichment of plasma membrane proteins for proteomic analysis. Multiple myeloma cells were grown and coated in suspension with three different pellicles of three different densities and both pellicle coated and uncoated suspensions analyzed by high-throughput LC-MS/MS. Enrichment was evaluated by the total number and the spectral counts of identified plasma membrane proteins

Enrichment of Plasma Membrane Proteins Using Nanoparticle Pellicles: Comparison between Silica and Higher Density Nanoparticles

Proteomic and other characterization of plasma membrane proteins is made difficult by their low abundance, hydrophobicity, frequent carboxylation, and dynamic population. We and others have proposed that underrepresentation in LC-MS/MS analysis can be partially compensated by enriching the plasma membrane and its proteins using cationic nanoparticle pellicles. The nanoparticles increase the density of plasma membrane sheets and thus enhance separation by centrifugation from other lysed cellular components. Herein, we test the hypothesis that the use of nanoparticles with increased densities can provide enhanced enrichment of plasma membrane proteins for proteomic analysis. Multiple myeloma cells were grown and coated in suspension with three different pellicles of three different densities and both pellicle coated and uncoated suspensions analyzed by high-throughput LC-MS/MS. Enrichment was evaluated by the total number and the spectral counts of identified plasma membrane proteins

UVnovo: A <i>de Novo</i> Sequencing Algorithm Using Single Series of Fragment Ions via Chromophore Tagging and 351 nm Ultraviolet Photodissociation Mass Spectrometry

<i>De novo</i> peptide sequencing by mass spectrometry represents an important strategy for characterizing novel peptides and proteins, in which a peptide’s amino acid sequence is inferred directly from the precursor peptide mass and tandem mass spectrum (MS/MS or MS³) fragment ions, without comparison to a reference proteome. This method is ideal for organisms or samples lacking a complete or well-annotated reference sequence set. One of the major barriers to <i>de novo</i> spectral interpretation arises from confusion of N- and C-terminal ion series due to the symmetry between <i>b</i> and <i>y</i> ion pairs created by collisional activation methods (or <i>c</i>, <i>z</i> ions for electron-based activation methods). This is known as the “antisymmetric path problem” and leads to inverted amino acid subsequences within a <i>de novo</i> reconstruction. Here, we combine several key strategies for <i>de novo</i> peptide sequencing into a single high-throughput pipeline: high-efficiency carbamylation blocks lysine side chains, and subsequent tryptic digestion and N-terminal peptide derivatization with the ultraviolet chromophore AMCA yield peptides susceptible to 351 nm ultraviolet photodissociation (UVPD). UVPD-MS/MS of the AMCA-modified peptides then predominantly produces <i>y</i> ions in the MS/MS spectra, specifically addressing the antisymmetric path problem. Finally, the program UVnovo applies a random forest algorithm to automatically learn from and then interpret UVPD mass spectra, passing results to a hidden Markov model for <i>de novo</i> sequence prediction and scoring. We show this combined strategy provides high-performance <i>de novo</i> peptide sequencing, enabling the <i>de novo</i> sequencing of thousands of peptides from an <i>Escherichia coli</i> lysate at high confidence