Improving Peptide Fragmentation for Hydrogen-Deuterium Exchange Mass Spectrometry Using a Time-Dependent Collision Energy Calculator.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is becoming a popular technique for interrogating biological systems. In recent years, advancements have been made to increase peptide coverage for proteins that resist digestion such as antibodies and membrane proteins. These methods commonly include using alternative digestion enzymes or longer chromatographic gradients, which may be expensive or time-consuming to implement. Here, we recommend an efficient proteomics-based approach to increase peptide confidence and coverage. A major filtering parameter for peptides in HDX is the number of product ions detected; this is a result of the collision energy (CE) applied within the MS. A traditional linear ramp achieves optimal CE for only short periods of time. More product ions will be created and detected if optimal CE can be achieved for a longer period of time. As a result, the coverage, redundancy, and data confidence are all increased. We achieved this by implementing a mobility-dependent CE look up table (LUT) which increases the CE as a function of mobility. We developed a program to calculate the optimal CE for a set of peptides and MS settings based on initial reference samples. We demonstrated the utility of the CE LUT on three protein samples including the soluble phosphorylase B, IgG2, and the membrane-stabilized AcrB. We showed that applying a CE LUT provided 8.5-50% more peptides compared to a linear CE ramp. The results demonstrate that a time-dependent CE LUT is a quick and inexpensive method to increase data confidence and peptide abundance for HDX-MS experiments.

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 µg of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Surface induced dissociation yields quaternary substructure of refractory noncovalent phosphorylase B and glutamate dehydrogenase complexes.

Ion mobility (IM) and tandem mass spectrometry (MS/MS) coupled with native MS are useful for studying noncovalent protein complexes. Collision induced dissociation (CID) is the most common MS/MS dissociation method. However, some protein complexes, including glycogen phosphorylase B kinase (PHB) and L-glutamate dehydrogenase (GDH) examined in this study, are resistant to dissociation by CID at the maximum collision energy available in the instrument. Surface induced dissociation (SID) was applied to dissociate the two refractory protein complexes. Different charge state precursor ions of the two complexes were examined by CID and SID. The PHB dimer was successfully dissociated to monomers and the GDH hexamer formed trimeric subcomplexes that are informative of its quaternary structure. The unfolding of the precursor and the percentages of the distinct products suggest that the dissociation pathways vary for different charge states. The precursors at lower charge states (+21 for PHB dimer and +27 for GDH hexamer) produce a higher percentage of folded fragments and dissociate more symmetrically than the precusors at higher charge states (+29 for PHB dimer and +39 for GDH hexamer). The precursors at lower charge state may be more native-like than the higher charge state because a higher percentage of folded fragments and a lower percentage of highly charged unfolded fragments are detected. The combination of SID and charge reduction is shown to be a powerful tool for quaternary structure analysis of refractory noncovalent protein complexes, as illustrated by the data for PHB dimer and GDH hexamer.

From protein complexes to subunit backbone fragments: a multi-stage approach to native mass spectrometry.

Native mass spectrometry (MS) is becoming an important integral part of structural proteomics and system biology research. The approach holds great promise for elucidating higher levels of protein structure: from primary to quaternary. This requires the most efficient use of tandem MS, which is the cornerstone of MS-based approaches. In this work, we advance a two-step fragmentation approach, or (pseudo)-MS(3), from native protein complexes to a set of constituent fragment ions. Using an efficient desolvation approach and quadrupole selection in the extended mass-to-charge (m/z) range, we have accomplished sequential dissociation of large protein complexes, such as phosporylase B (194 kDa), pyruvate kinase (232 kDa), and GroEL (801 kDa), to highly charged monomers which were then dissociated to a set of multiply charged fragmentation products. Fragment ion signals were acquired with a high resolution, high mass accuracy Orbitrap instrument that enabled highly confident identifications of the precursor monomer subunits. The developed approach is expected to enable characterization of stoichiometry and composition of endogenous native protein complexes at an unprecedented level of detail.

Effect of crowding and chaperones on self-association, aggregation and reconstitution of apophosphorylase b.

It was shown that the rate of reconstruction of muscle glycogen phosphorylase b (Phb) from apoenzyme and pyridoxal 5'-phosphate decreased under crowding conditions. The effect of crowding was counteracted by chaperones (α-crystallin and proline). Sedimentation analysis shows that crowding stimulates the formation of high-molecular-weight associates at 25 °C, whereas chaperones stabilize small oligomers. The study of the kinetics of apoPhb aggregation at 37 °C showed that the anti-aggregation activity of chaperones decreased under crowding conditions. When studying the sedimentation behavior of the mixture of apoPhb and α-crystallin, the complexes between unfolded apoPhb and dissociated forms of α-crystallin were observed. It is assumed that these complexes are responsible for realization of the chaperone-like activity of α-crystallin under crowding conditions.

Synthesis of branched polysaccharides with tunable degree of branching.

An in vitro enzyme-catalyzed tandem reaction using the enzymes phosphorylase b from rabbit muscle and Deinococcus geothermalis glycogen branching enzyme (Dg GBE) to obtain branched polyglucans with tunable degree of branching (2% ÷ 13%) is presented. The tunable degree of branching is obtained by varying the reaction conditions such as pH value, the choice of reducing agent and its concentration and reaction time. Linear amylose is formed by the phosphorylase-catalyzed propagation of glucose-1-phosphate while Dg GBE introduces branching points on the α-(1→6) position by relocating short oligosaccharide chains. Our results show that the best way to obtain different degrees of branching with this set of enzymes is by regulation of the reaction time.

Determination of ¹5N-incorporation into plant proteins and their absolute quantitation: a new tool to study nitrogen flux dynamics and protein pool sizes elicited by plant-herbivore interactions.

Herbivory leads to changes in the allocation of nitrogen among different pools and tissues; however, a detailed quantitative analysis of these changes has been lacking. Here, we demonstrate that a mass spectrometric data-independent acquisition approach known as LC-MS(E), combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and (15)N flux in a high throughput manner. The reliable identification/quantitation of rabbit phosphorylase b protein spiked into leaf protein extract was achieved. The linear dynamic range, reproducibility of technical and biological replicates, and differences between measured and expected (15)N-incorporation into the small (SSU) and large (LSU) subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO) and RuBisCO activase 2 (RCA2) of Nicotiana attenuata plants grown in hydroponic culture at different known concentrations of (15)N-labeled nitrate were used to further evaluate the procedure. The utility of the method for whole-plant studies in ecologically realistic contexts was demonstrated by using (15)N-pulse protocols on plants growing in soil under unknown (15)N-incorporation levels. Additionally, we quantified the amounts of lipoxygenase 2 (LOX2) protein, an enzyme important in antiherbivore defense responses, demonstrating that the approach allows for in-depth quantitative proteomics and (15)N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.

Alkylated dihydroxybenzoic acid as a MALDI matrix additive for hydrophobic peptide analysis.

Hydrophobic peptides are generally difficult to detect using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) because the majority of MALDI matrixes are hydrophilic and therefore have a low affinity for hydrophobic peptides. Here, we report on a novel matrix additive, o-alkylated dihydroxybenzoic acid (ADHB), which is a 2,5-dihydroxybenzoic acid (DHB) derivative incorporating a hydrophobic alkyl chain on a hydroxyl group to improve its affinity for hydrophobic peptides, thereby improving MALDI-MS sensitivity. The addition of ADHB to the conventional matrix α-cyano-4-hydroxycinnamic acid (CHCA) improved the sensitivity of hydrophobic peptides 10- to 100-fold. The sequence coverage of phosphorylase b digest was increased using ADHB. MS imaging indicated that hydrophobic peptides were enriched in the rim of a matrix/analyte dried spot when ADHB was used. In conclusion, the addition of ADHB to the standard matrix led to improved sensitivity of hydrophobic peptides by MALDI-MS.

A protein aggregation based test for screening of the agents affecting thermostability of proteins.

To search for agents affecting thermal stability of proteins, a test based on the registration of protein aggregation in the regime of heating with a constant rate was used. The initial parts of the dependences of the light scattering intensity (I) on temperature (T) were analyzed using the following empiric equation: I = K(agg)(T-T(0))(2), where K(agg) is the parameter characterizing the initial rate of aggregation and T(0) is a temperature at which the initial increase in the light scattering intensity is registered. The aggregation data are interpreted in the frame of the model assuming the formation of the start aggregates at the initial stages of the aggregation process. Parameter T(0) corresponds to the moment of the origination of the start aggregates. The applicability of the proposed approach was demonstrated on the examples of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscles and bovine liver glutamate dehydrogenase studied in the presence of agents of different chemical nature. The elaborated approach to the study of protein aggregation may be used for rapid identification of small molecules that interact with protein targets.

A method for N-terminal de novo sequencing of Nα-blocked proteins by mass spectrometry.

A method for de novo sequencing of N(α)-blocked proteins by mass spectrometry (MS) is presented. The approach consists of enzymatic digestion of N(α)-blocked protein, recovery of N-terminal peptide by depletion of non-N-terminal peptides from the digest pool, and selective derivatization of a C-terminal α-carboxyl group of isolated N-terminal peptide. The C-terminal α-carboxyl group of the N-terminal peptide was selectively derivatized with 3-aminopropyl-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine), according to oxazolone chemistry. The reagent TMPP-propylamine was designed to facilitate sequence analysis with MALDI-MS by mass- and charge-tagging. All of the identities and N-terminal sequences of two N(α)-acetylated proteins (rabbit phosphorylase b and bovine calmodulin) and human orexin A, which has pyroglutamic acid at the N-terminus, were successfully analyzed by allowing for the y-type ions almost exclusively.

Ion mobility-mass spectrometry of phosphorylase B ions generated with supercharging reagents but in charge-reducing buffer.

We investigate whether "supercharging" reagents able to shift the charge state distributions (CSDs) of electrosprayed protein ions upward also influence gas-phase protein structure. A differential mobility analyzer and a mass spectrometer are combined in series (DMA-MS) to measure the mass and mobility of monomer and multimeric phosphorylase B ions (monomer molecular weight ∼97 kDa) in atmospheric pressure air. Proteins are electrosprayed from charge-reducing triethylammonium formate in water (pH = 6.8) with and without the addition of the supercharging reagent tetramethylene sulfone (sulfolane). Because the DMA measures ion mobility prior to collisional heating or declustering, it probes the structure of supercharged protein ions immediately following solvent (water) evaporation. As in prior studies, the addition of sulfolane is found to drastically increase both the mean and maximum charge state of phosphorylase B ions. Ions from all protein n-mers were found to yield mobilities that, for a given charge state, were ∼6-10% higher in the absence of sulfolane. We find that the mobility decrease which arises with sulfolane is substantially smaller than that typically observed for folded-to-unfolded transitions in protein ions (where a ∼60% decrease in mobility is typical), suggesting that supercharging reagents do not cause structural protein modifications in solution as large as noted recently by Williams and colleagues [E. R. Williams et al., J. Am. Soc. Mass Spectrom., 2010, 21, 1762-1774]. In fact, the measurements described here indicate that the modest mobility decrease observed can be partly attributed to sulfolane trapping within the protein ions during DMA measurements, and probably also in solution. As the most abundant peaks in measured mass-mobility spectra for ions produced with and without sulfolane correspond to non-covalently bound phosphorylase B dimers, we find that in spite of a change in mobility/cross section, sulfolane addition does not substantially alter the structure of non-covalently bound protein complexes in the gas-phase.

1-(3-Deoxy-3-fluoro-beta-d-glucopyranosyl) pyrimidine derivatives as inhibitors of glycogen phosphorylase b: Kinetic, crystallographic and modelling studies.

Design of inhibitors of glycogen phosphorylase (GP) with pharmaceutical applications in improving glycaemic control in type 2 diabetes is a promising therapeutic strategy. The catalytic site of muscle glycogen phosphorylase b (GPb) has been probed with five deoxy-fluro-glucose derivatives. These inhibitors had fluorine instead of hydroxyl at the 3' position of the glucose moiety and a variety of pyrimidine derivatives at the 1' position. The best of this carbohydrate-based family of five inhibitors displays a K(i) value of 46muM. To elucidate the mechanism of inhibition for these compounds, the crystal structures of GPb in complex with each ligand were determined and refined to high resolution. The structures demonstrated that the inhibitors bind preferentially at the catalytic site and promote the less active T state conformation of the enzyme by making several favorable contacts with residues of the 280s loop. Fluorine is engaged in hydrogen bond interactions but does not improve glucose potency. The pyrimidine groups are located between residues 284-286 of the 280s loop, Ala383 of the 380s loop, and His341 of the beta-pocket. These interactions appear important in stabilizing the inactive quaternary T state of the enzyme. As a follow up to recent computations performed on beta-d-glucose pyrimidine derivatives, tautomeric forms of ligands 1-5 were considered as potential binding states. Using Glide-XP docking and QM/MM calculations, the ligands 2 and 5 are predicted to bind in different tautomeric states in their respective GPb complexes. Also, using alpha-d-glucose as a benchmark model, a series of substitutions for glucose -OH at the 3' (equatorial) position were investigated for their potential to improve the binding affinity of glucose-based GPb catalytic site inhibitors. Glide-XP and quantum mechanics polarized ligand (QPLD-SP/XP) docking calculations revealed favorable binding at this position to be dominated by hydrogen bond contributions; none of the substitutions (including fluorine) out-performed the native -OH substituent which can act both as hydrogen bond donor and acceptor. The structural analyses of these compounds can be exploited towards the development of better inhibitors.

An adsorption-based model for pulse duration in resistive-pulse protein sensing.

We have been investigating an electrochemical single-molecule counting experiment called nanopore resistive-pulse sensing. The sensor element is a conically shaped gold nanotube embedded in a thin polymeric membrane. We have been especially interested in counting protein molecules using these nanotube sensors. This is accomplished by placing the nanotube membrane between two electrolyte solutions, applying a transmembrane potential difference, and measuring the resulting ionic current flowing through the nanopore. In simplest terms, when a protein molecule enters and translocates the nanopore, it transiently blocks the ion current, resulting in a downward current pulse. We have found that the duration of such current-pulses are many orders of magnitude longer than the electrophoretic transport time of the protein through the nanotube detection zone. We develop here a simple model that accounts for this key, and previously explained, observation. This model assumes that the protein molecule engages in repeated adsorption/desorption events to/from the nanotube walls as it translocates through the detection zone. This model not only accounts for the long pulse duration but also for the triangular shape of the current pulse and the increase in the standard deviation of the pulse duration with increasing protein size. Furthermore, the results of our analyses are in general agreement with results obtained from other investigations of protein adsorption to surfaces. This includes the observations that smaller proteins stick more readily to the surface but remain adsorbed for shorter times than larger proteins. In addition, the sticking probabilities calculated from our data are in general agreement with results obtained from other methods.

Enzyme-responsive artificial chaperone system with amphiphilic amylose primer.

An enzyme-responsive artificial chaperone system which employs an amphiphilic amylose primer (dodecyl maltopentaose, C12-MP) as a surfactant and phosphorylase b was designed to enable protein refolding. Effective refolding of carbonic anhydrase B after both heat denaturation (70 degrees C for 10min) and guanidine hydrochloride (6M) denaturation was observed by controlled association between the protein molecules and the C12-MP primer micelle through an enzymatic reaction.

A fused silica micro-electrospray tip with an electrically floating metal wire insert to achieve more stable electrospray ionization.

A new electrospray tip with a wire insert was tested and compared with the conventional bare fused silica capillary tip. The new tip combined the approach of conventional fused silica spray tips with those containing metal wires. Here, we used a floating wire so that the tips could be prepared and replaced more easily. With the conventional tip, the electrospray process became unstable and the spray current fluctuated significantly in the presence of an air bubble. When the wire-inserted tip was used under the same conditions, much less signal deterioration occurred. The superior performance of this tip over the conventional tip was attributable to its enhanced electric conduction. The new tip has great potential for improving signal stability in LC mass spectrometry.

Effect of alpha-crystallin on thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscle.

Thermal aggregation of rabbit skeletal muscle glycogen phosphorylase b (Phb) has been investigated using dynamic light scattering under conditions of a constant rate of temperature increase (1 K/min). The linear behavior of the dependence of the hydrodynamic radius on temperature for Phb aggregation is consistent with the idea that thermal aggregation of proteins proceeds in the kinetic regime wherein the rate of aggregation is limited by diffusion of the interacting particles (the regime of "diffusion-limited cluster-cluster aggregation"). In the presence of alpha-crystallin, a protein exhibiting chaperone-like activity, the dependence of the hydrodynamic radius on temperature follows the exponential law; this suggests that the aggregation process proceeds in the kinetic regime where the sticking probability for colliding particles becomes lower than unity (the regime of "reaction-limited cluster-cluster aggregation"). Based on analysis of the ratio between the light scattering intensity and the hydrodynamic radius of Phb aggregates, it has been concluded that the addition of alpha-crystallin results in formation of smaller size starting aggregates. The data on differential scanning calorimetry indicate that alpha-crystallin interacts with the intermediates of the unfolding process of the Phb molecule. The proposed scheme of thermal denaturation and aggregation of Phb includes the stage of reversible dissociation of dimers of Phb into monomers, the stage of the formation of the starting aggregates from the denatured monomers of Phb, and the stage of the sticking of the starting aggregates and higher order aggregates. Dissociation of Phb dimer into monomers at elevated temperatures has been confirmed by analytical ultracentrifugation.

Effect of molecular crowding on the enzymes of glycogenolysis.

Cell cytoplasm contains high concentrations of macromolecules occupying a significant part of the cell volume (crowding conditions). According to modern concepts, crowding has a pronounced effect on the rate and equilibrium of biochemical reactions and stimulates the formation of more compact structures. This review considers different aspects of the crowding effect in vivo and in vitro, its role in regulation of cell volume, the effect of crowding on various interactions, such as protein-ligand and protein-protein interactions, as well as on protein denaturation, conformation transitions of macromolecules, and supramolecular structure formation. The influence of crowding arising from the presence of high concentrations of osmolytes on the interactions of the enzymes of glycogenolysis has been demonstrated. It has been established that, in accordance with predictions of crowding theory, trimethylamine N-oxide (TMAO) and betaine highly stimulate the association of phosphorylase kinase (PhK) and its interaction with glycogen. However, high concentrations of proline, betaine, and TMAO completely suppress the formation of PhK complex with phosphorylase b (Phb). The protective effect of osmolyte-induced molecular crowding on Phb denaturation by guanidine hydrochloride is shown. The influence of crowding on the interaction of Phb with allosteric inhibitor FAD has been revealed. The results show that, under crowding conditions, the equilibrium of the isomerization of Phb shifts towards a more compact dimeric state with decreased affinity for FAD.

2,5-Dihydroxyacetophenone: a matrix for highly sensitive matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of proteins using manual and automated preparation techniques.

2,5-Dihydroxyacetophenone (DHAP) is presented as a matrix which enables highly sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of peptides, proteins and glycoproteins on AnchorChip targets. Depending on the protein, lower fmol amounts can be detected due to the increased homogeneity and concentration of the crystallization of the analyte/matrix mixture on the anchors. Best results could be generated in the mass range of 8-100 kDa. All sample/matrix preparation steps starting from mixing of DHAP matrix solution with sample solution to the transfer of the mixture to the MALDI-TOF target can be performed manually or automatically allowing low- and high-throughput analyses.

Characterization of phospholipid-protein interactions by capillary isoelectric focusing with whole-column imaging detection.

The integration of functional proteins in the phospholipid bilayer is one of the most crucial features of biological membrane architecture. Phospholipid-protein interactions play an important role in the functions of bounded proteins in the phospholipid membrane. When the phospholipid-protein interactions occur, the protein structure tends to alter, which can result in a change in the isoelectric points (pI) of protein. Capillary isoelectric focusing (cIEF) with whole-column imaging detection (WCID) is an attractive technique that has the features of simple operation, high resolution, and fast separation without focused band mobility for detection of amphoteric biomolecules. In this study, a cIEF-WCID method was developed to characterize the phospholipids-protein interactions by monitoring the protein cIEF profiles. Seven proteins with different pI and molecular mass , and a zwitterionic phosphatidylcholine (PC) with zwitterionic properties, were used to evaluate the feasibility of the cIEF-WCID approach in the study of phospholipid-protein interactions. The cIEF profiles changed in response to the changes in protein conformation, clearly exhibiting interactions between the PC vesicles and the targeted proteins. The formation of PC-protein complex was observed in the cIEF electropherograms. It was demonstrated that seven proteins displayed distinct interactions with the PC vesicles due to their different chemical and physical properties. The influences of the PC concentration, incubation time, and incubation temperature on the phospholipids-protein interactions were investigated. This study validated a novel analytical approach for the characterization of phospholipid-protein interactions.

Dynamic process of phospholipid-protein interaction studied by capillary isoelectric focusing with whole-column imaging detection.

Liposomes are vesicles formed by the aggregation of amphiphilic phospholipid molecules, which can mimic natural cell membranes. Interaction between liposome and protein is important for the structure and function of natural cell membranes. In this study, a CIEF method with whole-column imaging detection was developed for monitoring the dynamic process of phospholipid-protein interactions. The CIEF profiles at successive interaction times clearly displayed the formation of the different conjugates between phospholipid and protein at different stages. Due to the diversity of the chemical and physical properties of targeted proteins in this study (trypsin inhibitor, beta-lactoglobulin B, phosphorylase b, and trypsinogen), different dynamic processes of phospholipid-protein interactions were exhibited. The type of phospholipids played an important role in the dynamic process of phospholipid-protein interaction, as noted by the use of zwitterionic phospholipid (phosphatidylcholine) and acidic phospholipid (phosphatidylserine). Mechanisms involved in these interactions were discussed by monitoring the dynamic processes in this study.