Protein sequencing data acquisition

The former is a protein of 14.7 kDa involved in the multienzyme nucleotide excision repair (NER) pathway with a determined NMR solution structure . In this protein, the Zn + possesses rather a structural than a catalytic role. Zn NMR spectra were acquired using a rather sophisticated probe (for details, see Reference 87) and operating at temperatures 5-250 K. Data acquisition was performed with the application of spin-echo methods for enhanced sensitivity . Specifically, experiments were carried out at 25 K using a combination of CP (cross-polarization) and spikelet echo pulse sequences which provide a considerable increase in signal-to-noise ratio (of the order of 30) relative to a classical quadrupole echo pulse sequence. The proton field strength applied to the above measurements was 60 kHz with a matching field of 20 kHz for zinc and a contact time... 

In our AFM-FS experiments of protein-DNA complexes, the respective DNA target sequences were covalently coupled to Si3N4 AFM cantilevers via monolayer activation and a functional heterobifunctional spacer molecule. The corresponding (His)6ExpG protein was immobilized on a silanized mica surfaces by a short linker molecule coupled to one of the five ExpG lysines. We used a commercial AFM instrument at 25°C that has been modified with a home-built electronics and data acquisition (Bartels et al. 2003). 

Sequencing was done in ABI protein sequencer models 470,477, or 473 using Blott Cartridges and using standard cycles and conditions for sequencing from PVDF membranes. Data acquisition and analysis was done using ABI 610 software. 

White et al. [53] reported open-access LC-MS cf. Ch. 9.2) for the rapid characterization of proteins. The system allows web-based sample submission and registration, automated data processing, interpretation, and report generation. The submitted amino-acid sequence is used to set parameters for the transformation of the mass spectmm into the protein molecular weight and to evaluate whether the submitted protein is actually detected. Data acquisition is performed on a TOF instrument in order to achieve sufficient mass accuracy. 

Data-dependent switching between a survey-MS mode and the product-ion MS-MS mode (Ch. 2.4.2) in the LC-MS analysis of tryptic digest on a triple-quadrapole instrument was pioneered by Stahl et al. [140]. The MS-MS spectra obtained were correlated wiA a protein sequence database by using the SEQUEST program. DDA (also called SmartSelect, or Information-Dependent Acquisition, IDA) on ion-trap, Q-LIT, and Q-TOF instruments have become important tools in high-throughput protein characterization. 

T-90°-data acquisition [9] in which the time interval, x, is chosen such that the water resonance, which is expected to have the longest T, in the sample, has zero magnetization after the 90° pulse [113, 114], The CPMG spin-echo pulse sequence 90°-(t-180°-t) n-data acquisition (n = number of repetitions), has been used with the pulse interval, r, adjusted to attenuate the water signal, for example, from erythrocyte and protein suspensions [113], The technique is improved by the addition of ionic species such as ammonium chloride which increases the chemical exchange of the water protons and thus shortens T2 relative to the compounds of interest. This method is known as WATR (Water Attenuation by T2 Relaxation) [114]. Solvent suppression can also be achieved by selective excitation of the spectrum with special pulses such that the water resonance occurs at a point of null excitation [115-119]. However, distortion of peaks near the null point may occur. 

An important factor in the progress of bioinformatics has been the constant increase in computer speed and memory capacity of desktop computers and the increasing sophistication of data processing techniques. The computation power of common personal computers has increased within 12 years approximately 100-fold in processor speed, 250-fold in RAM memory space and 500-fold or more in hard disk space, while the price has nearly halved. This enables acquisition, transformation, visuahsation and interpretation of large amounts of data at a fraction of the cost compared to 12 years ago. Presently, bioanalytical databases are also growing quickly in size and many databases are directly accessible via the Internet One of the first chemical databases to be placed on the Internet was the Brookha-ven protein data bank, which contains very valuable three-dimensional structural data of proteins. The primary resource for proteomics is the ExPASy (Expert Protein Analysis System) database, which is dedicated to the analysis of protein sequences and structures and contains a rapidly growing index of 2D-gel electrophoresis maps. Some primary biomolecular database resources compiled from spectroscopic data are given in Tab. 14.1. 

Figure 12,4 Data workflow with statistical analysis. The data processing and analysis follows the following sequence (a) Acquisition of files for the protein in the apo state and bound with the ligands of interest over several time points, (b) Isotope measurement and %D determination, (c) Aggregate sample percent deuteratlon Is arrived at by the mean of the individual on-exchange time point mean values. The standard deviation for the sample Is the RMS error of each time point divided by the number of time points, (d) Perturbation %D = ligand aggregate %D-apo aggregate %D. Standard error is calculated by taking the root sum squared of aggregate sample standard deviations, (e) Results are subjected to t-test and Tukey comparison, (f) Visualization components represent the results. Reproduced from Ref. [1], with permission from Elsevier...

The fragmentation efficiency of a protein ion in the gas phase appears to be strongly dependent on its amino acid sequence. It may be necessary to acquire a large number of laser shots (e.g., 10,000-40,000) for protein ions with low fragmentation efficiency in order to improve MS/MS signal-to-noise ratio (S/N). The 4800 TOF-TOF utilizes the third harmonic (A, = 355 nm) of a YAG solid state laser, which has a repetition rate of 200 Hz and a pulse width of 5 ns. Forty thousand laser shots correspond to 3 min of data acquisition. Depending on the laser fluence used, it may be necessary to sum data collected from multiple MALDI spots. 

Selected entries from Methods in Enzymology [vol, page(s)] Acquisition of frequency-discriminated spectrum, 239, 162-166, 170 sensitivity, 239, 169-173 constant-time, 239, 23-26 doublequantum filtered, 239, 236 gradient pulse experiments, 239, 185-189 protein structural information, 239, 377-379 pulse sequence and coherence transfer pathway, 239, 148-149 paramagnetic metalloprotein, 239, 494-497 data recording, SWAT method, 239, 166-169, 172 line shapes, effects of gradient pulses, 239, 162-166 identification of protein amino acid resonances, 232, 100 cyclosporin A, 239, 240-241. 

Tryptic digests of RIP140 protein was subjected to LC-ESI-MS/MS. Three independent full-scan ion chromatograms from m/z 400-550, 550-750, and 750-1200 were recorded in an information-dependent acquisition (IDA) mode to acquire MS/MS data. The IDA data were searched online at MASCOT (http //www.matrixscience.com) MS/MS data search at the NCBI data bank. The MS/MS data were analyzed manually to confirm the sequence of the modified and the unmodified forms of the same peptide identified by the data bank search. The full scan chromatograms were analyzed to assign the charged state, retention time, and intensities of the peptides. 

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