New Protein Structure Analysis Means - Progress in Hydroquinone Exchange Mass Spectrometry

--- Jia Wei, Chen Xiwo Special Technology (Shanghai) Co., Ltd. Experimental Center

Hydroquinone exchange mass spectrometry is a mass spectrometry technique for studying the spatial conformation of proteins. It has a wide range of applications in protein structure and dynamics research, protein interaction site discovery, protein epitopes and active site identification. With the continuous development of hydroquinone exchange mass spectrometry, it is becoming an important means for structural biologists and biopharmaceutical research and development.

Hydrogen deuterium exchange mass spectrometry (HDX MS) is a mass spectrometry technique for studying the spatial conformation of proteins. The principle is that the protein is immersed in a heavy aqueous solution, the hydrogen atoms of the protein will be exchanged in the helium atom of the heavy water, and the hydrogen on the surface of the protein in close contact with the heavy water is exchanged faster than the hydrogen inside the protein or involved in the formation of hydrogen bonds. The rate of hydroquinone exchange of different sequence fragments of the protein was determined by mass spectrometry to obtain the spatial structure information of the protein [1]. This process is like immersing the fist in the water, then lifting the water and opening the palm of your hand. At this time, the wet back of the hand indicates that it is on the outer surface in the structure of the "fist", while the relatively dry palm indicates that it is the inside of the "fist". In addition to sample preparation, the main processes of hydroquinone exchange mass spectrometry include: exchange reaction, termination reaction, rapid digestion of protein into peptide, liquid phase separation, mass spectrometry detection, and data analysis. The exchange step needs to be carried out under a plurality of reaction time periods, such as 0s, 10s, 1min, 10min, 60min, etc., in order to draw an exchange rate curve, and obtain accurate and comprehensive information. Hydroquinone exchange mass spectrometry has a wide range of applications in protein structure and its dynamic changes [1], protein interaction sites [2], protein epitopes and active site identification [3].

Compared to classical protein structure research methods, such as X-ray crystallography and nuclear magnetic resonance (NMR), hydroquinone exchange mass spectrometry does not provide accurate protein spatial structure, it provides directly The main information includes which amino acid sequences are located at the surface positions of the protein's spatial structure (including dynamic changes), possible active sites, and protein-protein interaction sites. However, hydroquinone exchange mass spectrometry has advantages that other classical methods do not have: First, the study of dynamic changes in protein structure is a prominent advantage of hydroquinone exchange mass spectrometry, including active sites and epitopes in change; secondly, hydroquinone Exchange mass spectrometry also has unique advantages in the study of conformation of protein complexes. In addition, hydroquinone exchange mass spectrometry has a small requirement for samples and a relatively low purity requirement. The study object is the natural conformation of proteins in solution environment rather than crystal. Advantages such as medium conformation [1, 4, 5]. Since the publication of the first research paper in 1991, hydroquinone exchange mass spectrometry has been developed and has become a very important application field in structural biology and mass spectrometry [6]. However, the complex implementation of hydroquinone exchange mass spectrometry experiments has affected the extent of its application to some extent. The main difficulties are: 1. How to avoid the backcrossing phenomenon of the progeny peptide after exchange; 2. The high precision and reproducibility requirements of the experimental control; 3. How to accurately distinguish the superposed mass spectrum peaks caused by the exchange; Simple and efficient analysis software requirements; 5, the analysis of exchange sites in amino acid units. Since 2005, Waters has been continuously researching the above difficulties and has launched the only commercial fully automated hydroquinone exchange mass spectrometry system solution, nanoACQUITY UPLC ^{® .} HD-Exchange System (Figure 1). Worldwide, the system has helped scientists publish research papers in leading research journals including Cell and Nature [7, 8]. In addition to scientific research requirements, the Waters Hydrogen Exchange Mass Spectrometry System has also been recognized by many leading international pharmaceutical companies and used in the research of active sites and epitopes of protein drugs in the development of new drugs.

The backcrossing phenomenon in the hydroquinone exchange experiment will seriously affect the credibility of the experimental data and even lead to the occurrence of erroneous results. To avoid backcrossing, two things need to be done: minimize the liquid analysis time and ensure that the temperature and pH in the liquid quality analysis are the conditions required for the minimum backcross reaction coefficient. The Waters UPLC ^® system uses sub-nanometer chromatography particle packing, which has unparalleled resolution compared to the large particle packing used in HPLC. Therefore, UPLC can greatly shorten the liquid chromatography time requirement without losing the chromatographic separation effect [9]. For many years of engineering improvement in the temperature and pH control issues, the nanoACQUITY UPLC HD-Exchange System has achieved full control of the steps of digestion, liquid phase separation [10].

The requirement for accuracy and reproducibility of hydroquinone exchange mass spectrometry experiments is the second major difficulty in its application. In the experiment, it is generally necessary to collect data at multiple time points such as 0s, 10s, 1min, 10min, 60min, and 240min. If manual manual experiments are performed, it is difficult to perform precise operations at several time points such as 10S-10min. Taking into account the need for repeated experiments, manual manual operations will have an impact on the final data credibility. Moreover, the repeated and cumbersome experimental process will bring a lot of work pressure to the experimenter. The nanoACQUITY UPLC HD-Exchange System completely completes a series of experimental processes such as exchange, termination of exchange, injection, and enzymatic cutting through the intelligent robotic arm, and always guarantees different temperature environments required for each step. These automated processes not only ensure the reliability of the experimental data, improve the efficiency of the experiment, but also free the scientists from the tedious and repetitive experiments.

In the mass spectrometry data of the hydroquinone exchange experiment, as the exchange time is prolonged, the polypeptide of the exchange reaction occurs, and as the mass becomes larger, the mass spectrometry signal gradually shifts toward the high mass-to-charge ratio. Therefore, these mass spectral peaks may gradually overlap and overlap each other with the mass spectral peaks of the polypeptides that have not undergone exchange reaction. The mass spectrometry signals superimposed on each other not only affect the judgment of the peak assignment, but also increase the error of the exchange rate data. Because the exchange rate is judged by quantifying the peptides that are exchanged, there is no doubt that the superimposed and confusing mass spectral data will greatly affect the accurate quantification of the mass spectral peaks. This is completely incompetent for mass spectrometers that simply analyze the mass-to-charge ratio. However, this seemingly impossible task was overcome by the Waters nanoACQUITY UPLC HD-Exchange System. This is because, unlike other common mass spectrometers, Waters' SYNAPT ^® mass spectrometer platform also has the ability to separate according to ion size and morphology (walking wave ion mobility separation). In the data processing, in addition to the mass-to-charge ratio information of the polypeptide ions, different ions can be distinguished by the ion migration time (ion mobility dimension parameter). Therefore, this unique SYNPAT mass spectrometry technology named HDMSE can separate the overlapping polypeptides with the same mass-to-charge ratio, and easily solve the problem of mass spectral signal superposition and obtain accurate exchange rate data [11,12]. (figure 2). The SYNPAT mass spectrometer platform won the 2007 PITTCON Gold Award. It has introduced a new generation of SYNAPT G2HDMS, SYNAPT G2-S HDMS and other models, and has a variety of ion sources such as ESI and MALDI. In addition to hydroquinone exchange technology, SYNAPT mass spectrometry system is also unique in the study of protein complex structure, and many high-quality applications have been published [13,14,15].

The fourth key point in achieving hydroquinone exchange mass spectrometry is how to efficiently analyze the large amount of data generated by multiple time points and multiple iterations. Manually completing such a huge amount of information processing will consume a lot of time for scientists. The DynamX software from Waters Hydrogen Exchange Mass Spectrometry provides scientists with simple, intuitive results and a variety of presentations.

In some special studies, it is required to make accurate amino acid measurements on the protein hydroquinone exchange site, which is another difficulty in hydroquinone exchange mass spectrometry. The use of CID (collision-induced dissociation) fragmentation patterns in routine studies may result in the rearrangement of deuterium atoms within the polypeptide, resulting in inaccurate localization of the specific amino acids that are exchanged. The ETD (electron transfer dissociation) fragmentation mode provided by SYNPAT mass spectrometry can avoid information chaos caused by helium atom rearrangement and has good fragmentation signal [16].

Waters' nanoACQUITY UPLC HD-Exchange System provides an unprecedented and simple solution for hydroquinone exchange mass spectrometry experiments, which strongly promotes hydroquinone exchange technology in protein structure and dynamics research, protein interaction site discovery, and protein The use of epitopes and active site identification is becoming an indispensable work platform for many structural biology scientists and biopharmaceutical companies.

references

(1) John R. Engen, Analysis of Protein Conformation and Dynamics by Hydrogen/Deuterium Exchange MS. Anal. Chem. 2009,81, 7870–7875

(2) Engen et al. probing protein interactions using HD exchange ms in ms of protein interactions. Edited by Downard, John Wiley & Sons, Inc. 2007, 45-61

(3) Tiyanont K, Wales TE, Aste-Amezaga M, et al. Evidence for increased exposure of the Notch1 metalloprotease cleavage site upon conversion to an activated conformation. Structure. 2011, 19, 546-554

(4) Heck AJ. Native mass spectrometry: a bridge between interactomics and structural biology. Nat Methods. 2008, 5, 927-933.

(5) Esther van Duijn, Albert JR Heck. Mass spectrometric analysis of intact macromolecular chaperone complexes. Drug Discovery Today. Drug Discovery Today: Technologies Volume 3, 2006, 21-27

(6) Viswanat ham Katta, Brian T. C hait, Steven Ca r r. Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry. Rapid Commun. Mass Spectrom. 1991, 5, 214–217

(7) Chakraborty K, Chatila M, Sinha J, et al. Chaperonin-catalyzed rescue of kinetically trapped states in protein folding. Cell. 2010 Jul 9;142(1):112-22.

(8) Zhang J, Adrián FJ, Jahnke W, et al. Targeting Bcr-Abl by combining allosteric with AT P-binding-site inhibitors. Nature. 2010,463, 501-506

(9) Wu Y, Engen JR, Hobbins WB. Ultra performance liquid chromatography (UPLC) further improves hydrogen/deuterium exchange mass spectrometry. J Am Soc Mass Spectrom. 2006 , 17, 163-167

(10) Wales TE, Fadgen KE, Gerhardt GC, Engen JR. High-speed

And high-resolution UPLC separation at zero degrees Celsius. Anal Chem. 2008, 80, 6815-6820

(11) Giles K, Pringle SD, Worthington KR, et al. Applications of a travelling wave-based radio-frequency-only stacked ring ion guide. Rapid Commun Mass Spectrom. 2004, 18, 2401-2414

(12) Olivova P, C hen W, C ha kra borty AB, Gebler JC. Determination of N-glycosylation sites and site heterogeneity in a monoclonal antibody by electrospray quadrupole ion-mobility time-offlight mass spectrometry. Rapid Commun Mass Spectrom. 2008 , 22,29-40

(13) Ruotolo BT, Benesch JL, Sandercock AM, et al. Ion mobilitymass spectrometry analysis of large protein complexes. Nat Protoc. 2008, 3, 1139-52.

(14) Uetrecht C, Barbu IM, Shoemaker GK, et al. Interrogating viral capsid assembly with ion mobility-mass spectrometry. Nat Chem.2011, 3,126-132

(15) Bleiholder C, Dupuis NF, Wyttenbac h T, Bowers MT. Ion mobility-mass spectrometry reveals a conformational conversion from random assembly to β-sheet in amyloid fibril formation. Nat Chem.

2011, 3, 172-177

(16) Kasper D. Rand, Steven D. Pringle, Michael Morris, John R., et al. ETD in a Traveling Wave Ion Guide at Tuned Z-Spray Ion Source Conditions Allows for Site-Specific Hydrogen/Deuterium Exchange Measurements. J Am Soc Mass Spectrom. 2011, in press