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.

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.

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.

Dynamically multiplexed ion mobility time-of-flight mass spectrometry.

Ion mobility spectrometry-time-of-flight mass spectrometry (IMS-TOFMS) has been increasingly used in analysis of complex biological samples. A major challenge is to transform IMS-TOFMS to a high-sensitivity, high-throughput platform, for example, for proteomics applications. In this work, we have developed and integrated three advanced technologies, including efficient ion accumulation in an ion funnel trap prior to IMS separation, multiplexing (MP) of ion packet introduction into the IMS drift tube, and signal detection with an analog-to-digital converter, into the IMS-TOFMS system for the high-throughput analysis of highly complex proteolytic digests of, for example, blood plasma. To better address variable sample complexity, we have developed and rigorously evaluated a novel dynamic MP approach that ensures correlation of the analyzer performance with an ion source function and provides the improved dynamic range and sensitivity throughout the experiment. The MP IMS-TOFMS instrument has been shown to reliably detect peptides at a concentration of 1 nM in the presence of a highly complex matrix, as well as to provide a 3 orders of magnitude dynamic range and a mass measurement accuracy of better than 5 ppm. When matched against human blood plasma database, the detected IMS-TOF features were found to yield approximately 700 unique peptide identifications at a false discovery rate (FDR) of approximately 7.5%. Accounting for IMS information gave rise to a projected FDR of approximately 4%. Signal reproducibility was found to be greater than 80%, while the variations in the number of unique peptide identifications were <15%. A single sample analysis was completed in 15 min that constitutes almost 1 order of magnitude improvement compared to a more conventional LC-MS approach.

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.

Measurement of adenosine 3',5'-monophosphate-dependent protein kinase and phosphorylase activities in in vivo conditions.

Microassay procedures for cAMP-dependent protein kinase and phosphorylase were developed which detected these activities in less than 25 micrograms of frozen-dried epidermis from a punch biopsy of skin without homogenization. Using these procedures, the activation of cAMP-dependent protein kinase and phosphorylase by beta-adrenergic stimulation in mouse skin was studied in vivo. Cyclic AMP-dependent protein kinase was stimulated by isoproterenol and inhibited by propranolol. Isoproterenol stimulation also activated phosphorylase a in mouse skin. In normal epidermis and uninvolved and involved epidermis from psoriatic patients no significant differences were found in the activities of cAMP-dependent kinase and phosphorylase a. In all experiments we observed that the unstimulated activity ratios of phosphorylase a/total phosphorylase were around 20-30%; these values were much lower than those hitherto reported and show a preponderance of phosphorylase b rather than a. We suggest that in previous reports where phosphorylase a domination was found, phosphorylase b to a activation occurred during homogenization. The data also suggest that in the steady state no obvious defect in basic activities of cAMP-dependent protein kinase and phosphorylase is observed in psoriatic skin.

Turbidimetric method for determination of glycogen phosphorylase activity and its use for estimation of equilibrium position of enzymic reaction.

A turbidimetric method has been developed for the continuous monitoring of the enzyme reaction catalyzed by glycogen phosphorylase. This method is based on the registration of the turbidity of glycogen solution at wavelengths above 300 nm. It has been shown that the increase in the turbidity is strictly proportional to the quantity of glucose 1-phosphate formed during the enzyme reaction. The method has the advantage of continuity, and it is suitable for determining the initial rate of catalytic synthesis or degradation of glycogen in a relatively simple and fast way. The kinetic experiments may be carried out under various conditions. The method of calculation of the overall equilibrium constant of the enzyme reaction catalyzed by glycogen phosphorylase has been elaborated. This method is based on the analysis of the dependence of the initial rate of the enzymic reaction on the proportion of the substrate of the forward reaction: [Pi]/( [Pi] + [G-1-P] ).

Determination of metabolite and regulatory enzyme levels in Dirofilaria immitis and Ascaris suum: a comparative study.

Studies on metabolite levels in Dirofilaria immitis revealed similarities in several metabolites with those of Ascaris suum. The glycogen level in the filariid was however 3-4 times lower than that in A. suum. Levels of three regulatory enzymes were also determined in D. immitis and compared with those in A. suum. The activities of Hexokinase and Phosphofructokinase were similar. However, the levels of Glycogen phosphorylase b appeared to be much lower in the filariid than in A. suum. The subtle but important differences observed may reflect modifications of the parasite enzymes suggesting salient differences in the regulation of energy production from carbohydrates in the worms. The differences may also represent specialization required for the unique life style of the worms in their different locations in their hosts.

Comparative study of the conformational transitions of frog and rabbit phosphorylases B.

1. Conformational motility of the purified muscle glycogen phosphorylase B from two species of vertebrates (rabbit and frog) was investigated by the Hydrogen-Exchange method and Infrared Spectometry. 2. The experimental results of the 1H-2H exchange were expressed in terms of the probability P of exposure to isotopic solvent of phosphorylase peptide groups and in terms of the corresponding changes in standard free energy delta Go. 3. The combined methods used didn't show considerable differences of the protein conformations in the physiological pH region but rabbit phosphorylase was only characterized by rather more compact structure in comparison with frog phosphorylase.

Determination of glycogen and enzymes of glycogen metabolism in human hair follicles.

The skin epithelium and its organelles use glycogen as well as glucose as source of energy. Therefore the characterisation of glycogen metabolism and the enzymes involved is important in the study of mechanisms regulating the normal or abnormal differentiation of skin organelles such as sebaceous glands and hair follicles. The present paper describes fluorimetric methods for the determination of glycogen and for the measurements of phosphorylase and phosphorylase kinase activity in one and the same lysate of minute tissue samples. The methods were tested for their suitability on freshly isolated human hair follicles and cultured hair follicle cells. The possible use of these techniques for studies on the pathophysiology of acne and hirsutism is discussed.

Shift of glycolysis as a marker of sperm maturation.

The presence of glycogen-phosphorylase (A and B forms), glucose-6-phosphatase and fructose-1, 6-diphosphatase in bonnet monkey spermatozoa was determined. The process of glycolysis gradually decreased and the process of reversal of glycolysis steadily increased in monkey spermatozoa as they move from testis to caput to corpus to cauda to vas deferens and finally in ejaculate. Hence in bonnet monkeys, the functional maturity of spermatozoa and the shift in glycolysis are closely related.

A strategy for chemical sensing based on frequency tunable acoustic devices.

A new acoustic sensor geometry, the magnetic acoustic resonant sensor (MARS), is described. The device comprises a circular 0.5-mm-thick resonant plate fabricated from a wide variety of nonpiezoelectric materials and coated on the underside with a 2.5-microm-thick aluminum film. Harmonic radial shear waves over at least a 2 orders of magnitude frequency range can be induced in the resonant plate by enhanced magnetic direct generation using a noncontacting rf coil and NdFeB magnet. Mass loading with adherent aluminum films produced frequency changes of 106 Hz/nm (40.8 Hz/ng-mm(-2)), while contact with viscous fluids resulted in maximum changes of 15 446 Hz/cP. At an operating frequency of 50 MHz, the device detected viscosity changes as low as 0.0006 cP. The adsorption of proteins such as human IgG and the binding of a complementary antigen, goat anti-human IgG, on the upper nonmetallized surface of the device has been monitored with a detection limit of approximately 75 ng/mL. The binding of substrates and allosteric effectors to glycogen phosphorylase b has provided evidence that the device is very sensitive to viscoelastic changes in adsorbed proteins. The MARS device generates radial shear acoustic waves over a broad bandwidth that are unaffected by the conductivity of the solution. These results suggest that simple metal, glass, crystalline, or polycrystalline plates can be used as a new type of tunable acoustic immunosensor.

A continuous spectrophotometric method for the determination of glycogen phosphorylase-catalyzed reaction in the direction of glycogen synthesis.

We offer a "continuous" spectrophotometric method for the determination of the glycogen phosphorylase-catalyzed reaction in the direction of glycogen synthesis. This method relies on a coupled enzyme procedure, involving purine nucleoside phosphorylase and its chromophoric substrate, 2-amino-6-mercapto-7-methyl ribonucleoside (7-methyl-6-thioguanosine (MTGuo)), for the estimation of inorganic phosphate (M. R. Webb, Proc. Natl. Acad. Sci. USA 89, 4884-4887, 1992). We have examined the effects of the reaction components on the catalytic activities of both "primary" and "coupling" enzymes. While MTGuo exhibits no effect on the glycogen phosphorylase-catalyzed reaction, glucose 1-phosphate and AMP are partially inhibitory to nucleoside phosphorylase. However, the latter effects pose no problem as long as the coupling enzyme is maintained at a relatively higher concentration in the assay system. The coupled enzyme assay system, standardized for the measurement of glycogen phosphorlase activity, has enabled us to demonstrate explicitly that the rate of the enzyme-catalyzed reaction exhibits sigmoidal dependence on both AMP and glucose 1-phosphate concentrations. We argue that these sigmoidal profiles have been observed due to the sensitivity and precision of the present assay system.

[Morphological and biochemical studies on glycogenosis type V (McArdle) (author's transl)]. / Zur Morphologie und Biochemie der Glykogenose Typ V (McArdle).

This report deals with structural and biochemical studies of muscle biopsies from six patients with glycogenosis type V (McArdle). From a morphological point of view in four cases the typical findings of vacuolar myopathy with glycogen storage especially under the sarcolemma can be demonstrated. One biopsy shows only mild structural changes which without additional biochemical analysis could be overlooked. In one case signs of recovery phase after rhabdomyolysis predominate the storage myopathy. Biochemical studies in all cases show an elevated glycogen content (2.5-4.23%). Only the from a clinical point of view most expressive patient with recurrent episodes of rhabdomyolysis exhibits a glycogen storage over 5%. All cases additionally show an absence or highly reduction of phosphorylase activity. Apart from the most expressive clinical course the extent of morphological and biochemical findings is not clearly correlated. Therefore if clinical signs suggest the diagnosis of glycogenosis type V it appears necessary to perform additional biochemical examination of muscle biopsy independent from the degree of morphological anomalies.

Mixed-dye staining method for protein detection in polyacrylamide gel electrophoresis using calconcarboxylic acid and rhodamine B.

We have developed a new mixed-dye protein staining method that is simple, rapid, and sensitive. A freshly prepared mixture of calconcarboxylic acid (NN, 0.02%) and rhodamine B (RB, 0.04%) in 40% methanol/7% acetic acid, was used as a staining solution. RB acts as an auxiliary agent to inhibit the binding of NN to the gel matrix, reducing the background staining and therefore enhancing the protein staining by NN. This mixed-dye staining method reduces the total staining and destaining time to less than an hour, and increases the sensitivity to 25 ng of bovine serum albumin, which is greater than the 100 ng sensitivity limit of Coomassie Brilliant Blue R-250 (CBBR) staining.

Improved detection of higher molecular weight proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on polytetrafluoroethylene surfaces.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of proteins was performed on a range of polytetrafluoroethylene (PTFE) surfaces. Sinapinic acid and alpha-cyano-4-hydroxycinnamic acid matrices were compared and the order of application varied to identify the best combination for each surface. It is demonstrated that the use of a PTFE surface improves the intensity of signals obtained for higher molecular weight proteins.

High-performance capillary electrophoresis of proteins. Sodium dodecyl sulphate-polyacrylamide gel-filled capillary column for the determination of recombinant biotechnology-derived proteins.

Fused-silica capillary columns were filled with sodium dodecyl sulfate-polyacrylamide gel and the column effluent was monitored at 214 nm using a commercially available high-performance capillary electrophoresis (HPCE) instrument to separate and rapidly quantify recombinant biotechnology-derived proteins. An excellent linear relationship (r greater than 0.999) exists between the peak migration time and the molecular weights of reference proteins in the range 10,000-100,000 and 40,000-200,000 dalton by use of the capillary columns filled with acrylamide gel at a T composition of 5% and 3%, respectively. The relative standard deviation (R.S.D.) of the peak migration time is ca. 1%. Theoretical plates of 5 X 10(5)-1 X 10(6) per metre are routinely being obtained. Calibration graphs of peak area versus weight of recombinant biotechnology-derived proteins are linear (r greater than 0.999) and the proteins may be quantified with an R.S.D. of ca. 3-7%. As little as 50 nmol of a protein may be quantified and an impurity peak of molecular weight ca. 1500 less than that of the parent compound (ca. 60,000 dalton) may be differentiated by HPCE with a gel-filled capillary column.