Rachel R. Ogorzalek Loo

Enhanced FASP (eFASP) to increase proteome coverage and sample recovery for quantitative proteomic experiments.

The integrity of quantitative proteomic experiments depends on the reliability and the robustness of the protein extraction, solubilization, and digestion methods utilized. Combinations of detergents, chaotropes, and mechanical disruption can yield successful protein preparations; however, the methods subsequently required to eliminate these added contaminants, in addition to the salts, nucleic acids, and lipids already in the sample, can result in significant sample losses and incomplete contaminant removal. A recently introduced method for proteomic sample preparation, filter-aided sample preparation (FASP), cleverly circumvents many of the challenges associated with traditional protein purification methods but is associated with significant sample loss. Presented here is an enhanced FASP (eFASP) approach that incorporates alternative reagents to those of traditional FASP, improving sensitivity, recovery, and proteomic coverage for processed samples. The substitution of 0.2% deoxycholic acid for urea during eFASP digestion increases tryptic digestion efficiency for both cytosolic and membrane proteins yet obviates needed cleanup steps associated with use of the deoxycholate sodium salt. For classic FASP, prepassivating Microcon filter surfaces with 5% TWEEN-20 reduces peptide loss by 300%. An express eFASP method uses tris(2-carboxyethyl)phosphine and 4-vinylpyridine to alkylate proteins prior to deposition on the Microcon filter, increasing alkylation specificity and speeding processing.

Observation and Implications of High Mass-to-Charge Ratio Ions from Electrospray Ionization Mass Spectrometry

High mass-to-charge ratio ions (> 4000) from electrospray ionization (ESI) have been observed for several proteins, including bovine cytochrome c (Mr 12,231) and porcine pepsin (Mr 34,584), by using a quadrupole mass spectrometer with an m/z 45,000 range. The ESI mass spectrum for cytochrome c in an aqueous solution gives a charge state distribution that ranges from 12 + to 2 +, with a broad, low-intensity peak in the mass-to-charge ratio region corresponding to the [M + H]+ ion. the negative ion ESI mass spectrum for pepsin in 1% acetic acid solution shows a charge state distribution ranging from 7− to 2−. To observe the [M - H]− ion, harsher desolvation and interface conditions were required. Also observed was the abundant aggregation of the protens with average charge states substantially lower than observed for their monomeric counterparts. The negative ion ESI mass spectrum for cytochrome c in 1–100 mM NH4OAc solutions showed greater relative abundances for the higher mass-to-charge ratio ions than in acuidic solutions, with an [M - H]− ion relative abundance approximately 50% that of the most abundant charge state peak. The observation that protein aggregates are formed with charge states comparable to monomeric species (at fower mass-to-charge ratios) suggests that the high mass-to-charge ratio monomers may be formed by the dissociation of aggregate species. The observation of low charge state and aggregate molecular ions concurrently with highly charged species may serve to support a variation of the charged residue model, originally described by Dole and co-workers (Dole, M., et al. J. Chem. Phys.1968, 49, 2240; Mack, L. L., et al. J. Chem. Phys.1970, 52, 4977) which involves the Coulombically driven formation of either very highly solvated molecular ions or lower ananometer-diameter droplets.

A New Approach for the Study of Gas-Phase Ion-Ion Reactions Using Electrospray Ionization

A simple flow reactor which facilitates the study and application of ion-ion and ion-molecule reactions at near atmospheric pressures is reported. Reactant ions were generated by electrospray ionization and discharge ionization methods, although any ionization sources amenable to atmospheric pressure may be used. Ions of opposite charge are generated in spatially separate ion sources and are swept into capillary inlets where the flows are merged and where reaction(s) can occur. Among the reactions investigated were the partial neutralization of multiply protonated polypeptides and proteins such as melittin, bradykinin, cytochrome c, and myoglobin by reaction with discharge-generated anions, the partial neutralization of multiply charged anions of oligodeoxyadenylic acid (d(pA)3) by reaction with discharge-generated cations, the partial neutralization of bovine A-chain insulin anions by reaction with myoglobin [M+nH]n+ ions, and the reaction of multiply protonated melittin with discharge-generated cations. The cation-anion reactions generally resulted in a shift to lower charge (higher mass-to-charge ratio) in the products’ charge state distributions and the transfer of solvent molecules to the macromolecule products. Multiply protonated melittin was detected in a less highly solvated state with the positive discharge in operation.

Investigation of the gas-phase structure of electrosprayed proteins using ion-molecule reactions.

Proton transfer reactions of ammonia, dimemylamine, diethylamine, and trimethylarnine with multiply protonated proteins generated by electrospray ionization (ESI) were examined to probe the relationship between solution and gas-phase protein structure and the relationship with ion-molecule reactivity. The ion-molecule reactions were carried out in an atmospheric pressure capillary inlet/reactor based upon an ESI interface to a quadrupole mass spectrometer. Two types of systems were explored: (1) proteins possessing cysteine-cysteine disulfide bonds and the analogous disulfide-reduced proteins, and (2) proteins sprayed from solution compositions where the protein has different conformations. While the cysteine-cysteine disulfide-bound proteins were more reactive than equally charged disulfide-reduced proteins under these conditions, no significant reactivity differences were noted for ions arising from different solution conformations. The effect of inlet/reactor temperature on charge distributions with and without amine reagent was also explored, demonstrating that thermal denaturation of proteins can occur in heated capillary inlets. The results are discussed in the context of recent results indicating the persistence of at least some higher order protein structure in the gas phase.

Mass spectrometry of proteins directly from polyacrylamide gels

The direct combination of thin-layer gel electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry has been demonstrated with good sensitivity and mass accuracy, offering potential advantages in speed and reduced complexity. Mass spectra have been obtained from isoelectric focusing, sodium dodecyl sulfate, and native gels with as little as 660 fmol of α- and β-chain bovine hemoglobin and 1 pmol of horse heart myoglobin loaded. CNBr digests were performed in situ, and the products were probed in-gel. Noncovalent complexes such as multimeric protein systems, enzyme inhibitor complexes, and protein-ligand complexes can also be characterized when gel electrophoresis is run under nondenaturing conditions. This approach shows promise for simplifying the interface between gel electrophoresis and mass spectrometry.

Amino Acid Position-specific Contributions to Amyloid β-Protein Oligomerization

Understanding the structural and assembly dynamics of the amyloid β-protein (Aβ) has direct relevance to the development of therapeutic agents for Alzheimer disease. To elucidate these dynamics, we combined scanning amino acid substitution with a method for quantitative determination of the Aβ oligomer frequency distribution, photo-induced cross-linking of unmodified proteins (PICUP), to perform “scanning PICUP.” Tyr, a reactive group in PICUP, was substituted at position 1, 10, 20, 30, or 40 (for Aβ40) or 42 (for Aβ42). The effects of these substitutions were probed using circular dichroism spectroscopy, thioflavin T binding, electron microscopy, PICUP, and mass spectrometry. All peptides displayed a random coil → α/β → β transition, but substitution-dependent alterations in assembly kinetics and conformer complexity were observed. Tyr1-substituted homologues of Aβ40 and Aβ42 assembled the slowest and yielded unusual patterns of oligomer bands in gel electrophoresis experiments, suggesting oligomer compaction had occurred. Consistent with this suggestion was the observation of relatively narrow [Tyr1]Aβ40 fibrils. Substitution of Aβ40 at the C terminus decreased the population conformational complexity and substantially extended the highest order of oligomers observed. This latter effect was observed in both Aβ40 and Aβ42 as the Tyr substitution position number increased. The ability of a single substitution (Tyr1) to alter Aβ assembly kinetics and the oligomer frequency distribution suggests that the N terminus is not a benign peptide segment, but rather that Aβ conformational dynamics and assembly are affected significantly by the competition between the N and C termini to form a stable complex with the central hydrophobic cluster.

High sensitivity mass spectrometric methods for obtaining intact molecular weights from gel-separated proteins

The molecular weight measurement of intact Escherichia coli proteins separated by isoelectric focusing‐immobilized pH gradient (IEF‐IPG) gels and analyzed by mass spectrometry is presented. Two methods are discussed: (i) electrospray ionization (ESI) mass spectrometry (MS) of extracted proteins, and (ii) matrix‐assisted laser desorption/ionization (MALDI)‐MS analysis directly from IEF‐IPG gels. Both ESI and MALDI methods yield sub‐picomole sensitivity and good mass measurement accuracy. The use of an array detector for ESI‐MS was essential to discriminate against contaminating background ions and to selectively detect high mass protein ions. MALDI‐MS offers high‐throughput analysis of one‐ and potentially two‐dimensional (2‐D) gels. The “virtual 2‐D” gel method with first‐dimensional IEF separation and the second dimension as molecular mass determination by MS, is a particularly promising method for protein analysis due to its ultra high sensitivity and correspondence to classical 2‐D gels. Further sensitivity enhancements for the MALDI‐MS method are provided by post acceleration detection optimized for high mass time‐of‐flight analysis.

Proton transfer reaction studies of multiply charged proteins in a high mass-to-charge ratio quadrupole mass spectrometer

Proton transfer reactions of multiply charged ions at high mass-to-charge ratios were explored with a low frequency quadrupole mass spectrometer. This instrument enabled a qualitative comparison of proton transfer reaction rates at low charge states for ions generated by electrospray ionization (ESI) from different solution conformations and for disulfide-linked versus disulfide-reduced protein ions. Proton transfer reactions that efficiently reduced the number of charges for ESI-generated ions to approximately the number of arginines in the polypeptide sequence were observed. No significant differences in gas-phase reaction rates were noted between different solution conformers. Differences in reaction rates between “native” and disulfide-reduced proteins were much smaller than those observed below m/z 2000 with lower proton affinity reagents or by using lower reagent concentrations. These smaller differences in reaction rates are thought to reflect the reduced electrostatic contributions from widely spaced charge sites and thus, the reduced sensitivi ty to an ion’s three-dimensional structure or U compactness.

APPLYING CHARGE DISCRIMINATION WITH ELECTROSPRAY IONIZATION-MASS SPECTROMETRY TO PROTEIN ANALYSES

Electrospray ionization with a magnetic sector mass spectrometer and scanning array detector has unique advantages for sensitive analyses of large biomolecules. The ability to discriminate against low charge state ions (smaller peptides, buffers and salts, background ions) allows for detection of more highly charged ions from proteins present at much lower concentration relative to the small ions from buffers and detergents present. Low femtomole detection limits can be achieved for proteins greater than 100 ku. The charge discrimination phenomenon is more pronounced for higher charged ions, and especially for large biomolecules. Although the charge distribution for the monomer (66 ku) and dimer (133 ku) species of bovine serum albumin overlap, both species can be ascertained readily in a mixture because the lower charged monomer ions have higher optimum microchannel plate voltages than the higher charged dimer ions. Protein-containing solutions can be analyzed directly by electrospray ionization—mass spectrometry (ESI-MS) with array detection, which eliminates time-consuming separation and sample cleanup procedures. For example, heme-containing proteins can be directly detected from ESI-MS of human blood (hemoglobin) as well as from raw meat juices (hemoglobin and myoglobin).

A study of the thermal denaturation of ribonuclease S by electrospray ionization mass spectrometry.

The thermal stability of ribonuclease S (RNase S), an enzymatically active noncovalent complex composed of a 2166-u peptide (S-peptide) and a 11,534-u protein (S-protein), was investigated by electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis ESI-MS (CE-ESI-MS). The intensities of peaks corresponding to the RNase S complex were inversely related to both the applied nozzle-skimmer (or capillary-skimmer) voltage bias in the atmosphere-vacuum interface and the temperature of the RNase S solution. By using a heated metal capillary-skimmer interface and a room temperature solution of RNase S, the intensities of RNase S molecular ion peaks were observed to decrease with increasing metal capillary temperature. Mass spectrometric studies with both the nozzle-skimmer and capillary-skimmer interface designs allowed determination of phenomenological enthalpies for dissociation of the RNase S complex in both solution and for the electrosprayed microdroplet-gas phase species. Intact RNase S complex could also be detected with CE-ESI-MS separations by using a 10-mM ammonium bicarbonate (pH 7.9) solution as the electrophoretic buffer. These studies provide new insights into the stability of multiply charged noncovalent complexes in the gas phase and the mass spectrometric conditions required for such studies, and suggest that information regarding solution properties can be obtained by ESI-MS.