- Finding the proverbial needle
Novel cross-linker helps to identify peptides and thus to characterize protein structures.
Investigators working in the field of proteomics are interested in studying the three-dimensional structures of proteins and the covalent connections in protein-protein complexes so that they can map the contact points when the proteins are folded, for example. Chemical cross-linking coupled with mass spectrometric analysis often is used to access this information.
Researchers have reported a novel IR Chromogenic cross-linker (IRCX) that enables them to distinguish between cross-linked and noncross-linked peptides, thus facilitating study of the three-dimensional structures of proteins. After the cross-linking procedure, the protein or protein-protein complex undergoes enzymatic digestion, yielding smaller, more manageable peptides. Liquid chromatography infrared multiphoton dissociation mass spectroscopy (LC-IRMPD-MS) is performed, and only those peptides containing cross-inkers will photodissociate after irradiation with a 10.6-μm laser. Thus the researchers can simply determine which peptides are cross-linked and which are not.
This well-established technique still presents analytical challenges, however. Typically, after the cross-linking procedure, the protein or protein-protein complex is enzymatically digested, producing smaller, more manageable peptides that investigators use to detect and reconstruct the contact points in the protein.
Unfortunately, noted Jennifer S. Brodbelt, a researcher with the University of Texas at Austin, digestion of the protein yields dozens of noncross-linked peptides that offer little useful information, virtually overwhelming the sparse population of cross-linked peptides of primary interest to investigators. “Trying to find these low-abundance peptides within a huge mixture of peptides is like looking for a needle in a haystack,” she said.
In an Analytical Chemistry paper published July 1, 2008, Brodbelt and colleagues at the university reported the development of a novel cross-linker that allows them to differentiate cross-linked from noncross-linked peptides. “We decided to give the diagnostic cross-linked peptides a special signature by using a chromogenic cross-linking agent,” she explained. “We use a CO2 laser to irradiate all the peptides in the mass spectrometer, and only those peptides that contain the chromogenic cross-linker will photodissociate. Thus, by monitoring the photodissociation of various peptides, we can easily determine which ones are cross-linked and which ones are not cross-linked.”
Previous studies had shown that the infrared multiphoton dissociation technique offers an effective means to activate and dissociate biomolecules in the gas phase and to selectively dissociate phosphopeptides in Fourier transform ion cyclotron resonance and quadruple ion trap mass spectrometers. The researchers therefore designed the cross-linker with a strong infrared chromophore: a phosphate group with high absorption at 10.6 μm, the wavelength of a continuous-wave CO2 laser.
They demonstrated the technique using a mock mixture of unmodified peptides and infrared cross-linked (both intramolecularly and intermolecularly) peptides, performing experiments on a modified linear ion trap mass spectrometer made by Thermo Scientific of San Jose, Calif., running Xcalibur version 2.2 software and outfitted with the standard electrospray ionization source. Photodissociation was achieved using a CO2 continuous-wave laser made by Synrad Inc. of Mukilteo, Wash., with axial transmission of 10.6-μm radiation through the back end of the vacuum chamber.
The researchers first demonstrated that only cross-linked or dead-end modified peptides show efficient photodissociation following 50 ms of irradiation, thus establishing that they could identify species of interest simply by comparing ion abundances obtained before and after irradiation. For a tryptic digest of cross-linked ubiquitin, they observed four cross-links and two dead-end modifications and clearly identified the locations of the cross-links.
Other groups have reported development of novel cross-linkers with which to identify these locations using mass spectrometric techniques — by incorporating isotope labels that yield peaks within the mass spectra, for example, or fluorophores that help distinguish between modified and unmodified proteins and peptides. The technique described in the Analytical Chemistry paper offers enhanced selectivity with respect to these, Brodbelt said, allowing selective identification of important cross-linked peptides “even amidst dozens of other uninformative, noncross-linked peptides.”
The technique could contribute to a number of applications — for example, determining the contact points of interacting proteins that are involved in large biological assemblies such as the spliceosome. This method could be used also to map low-resolution three-dimensional structures of a protein whose fold changes upon activation.
The researchers more recently have been working to develop an ultraviolet chromogenic cross-linking agent that will respond to ultraviolet rather than infrared photons. This strategy entails a two-step conjugation method in which each biomolecule is labeled individually by one of two different heterobifunctional linkers. The cross-linking reaction ultimately creates a bis-aryl hydrazone bond that links the two constituents, thus affording a chromophore that absorbs at 355 nm, the wavelength of a tripled Nd:YAG laser.
The chromogenic hydrazone moiety is not present in any of the dead-end products, thus further enhancing the selective identification of the targeted cross-linked ones. Moving to different chromophores offers greater flexibility in incorporating the active chromophores into various types of cross-linkers, as well as increased selectivity of photoactivation. This additional tool will expand the range of applications and the versatility of the method, Brodbelt said.
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