Proteomics 2013, 13, 3245–3246 3245DOI 10.1002/pmic.201300445

Finding the missing link: Disulfide-containing proteins

via a high-throughput proteomics approach

Ramzi J. Khairallah and Sakthivel Sadayappan

Health Sciences Division, Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL,USA

Top-down proteomics have recently started to gain attention as a novel method to provideinsight into the structure of proteins in their native state, specifically the number and locationof disulfide bridges. However, previous techniques still relied on complex and time-consumingprotein purification and reduction reactions to yield useful information. In this issue ofProteomics, Zhao et al. (high-throughput screening of disulfide-containing proteins in a complexmixture, Proteomics 2013, 13, 3256–3260) devise a clever and rapid method for high-throughputdetermination of disulfides in proteins via reduction by tris(2-carboxyethyl)phosphine. Theirwork provides the foundation necessary to undertake more complex experiments in biologicalsamples.

Keywords:

Disulfide / Glutathionylation / Mass spectrometry / Post-translational modification /Redox proteomics / Redox modifications

Received: October 7, 2013Revised: October 7, 2013

Accepted: October 15, 2013

As MS enters the mainstream, novel methods are being de-vised to diversify the experimental questions that can be an-swered. Protein structure has usually been investigated byX-ray crystallography and NMR, however, these techniquesrequires highly purified protein crystals that are not amend-able to high-throughput screening. Fortunately, MS tech-niques have proved to be a complimentary approach to pro-vide structural information about proteins. Indeed, precisedetermination of disulfide bonds between cysteines (Cys)is an essential first step to examine protein structure. Cur-rent MS methods to reveal disulfide bridges generally employbottom-up techniques, with proteolytic digestion and exten-

Correspondence: Dr. Sakthivel Sadayappan, Health SciencesDivision, Department of Cell and Molecular Physiology, LoyolaUniversity of Chicago, Maywood, IL 60153, USAE-mail: ssadayappan@lumc.eduFax: +708-216-6308

Abbreviations: BLG, �-lactoglobulin; Cys, cysteines; Cyt C, cy-tochrome C; Lyso, lysozyme; Q-TOF, quadrupole TOF; Redox,reduction-oxidation; RNase B, ribonuclease B; RT, retention time;TCEP, tris(2-carboxyethyl)phosphine; Ubi, ubiquitin

sive, difficult sample preparation such as reduction and alky-lation [1, 2]. These methods, while useful, do not allow forhigh-throughput analysis of complex samples and providean incomplete image of disulfide bonds depending on se-quence coverage of peptides recovered from digestion. Morerecently, top-down approaches have been used to provide acomprehensive analysis of disulfide bonds, albeit in purifiedprotein samples [3].

In this issue [4], Zhao et al. expand on their previous workin the characterization of disulfide-bonded proteins of humansalivary �-amylase and adapt a relatively simple LC techniquewith novel sample preparation to significantly enhance theirmethod [3]. The aim of this study was to be able to developa top-down LC/MS approach that allows for the rapid de-termination of disulfide bonds in complex protein mixture.This important improvement is necessary in order to un-dertake high-throughput screenings of disulfide bridges inproteins in conditions where the reduction-oxidation (redox)balance of the cell is altered [2, 5]. Indeed, several patholo-gies display increased ROS formation leading to modifiedCys reactivity, such as tissue following ischemia/reperfusion[6], Alzheimer‘s disease [7], oncogenesis [8], and muscular

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3246 R. J. Khairallah and S. Sadayappan Proteomics 2013, 13, 3245–3246

dystrophy [9]. Understanding the role of modified disulfidelinks in cellular degeneration might open new avenues fortherapeutic intervention.

The authors use a clever approach, tris(2-carboxyethyl)phosphine (TCEP) in combination withlow pH to reduce their protein mixture. This approach to re-duction has the advantage of preventing disulfide scramblingand precludes alkylation of free Cys residues. TCEP is alsocompatible with LC/MS applications and does not requireremoval prior to running the samples. Thus, by comparingthe molecular weights of a reduced and nonreduced protein,the number of the disulfide bonds can be inferred owing toan increase of 2 Da in mass per bridge. Interestingly, theauthors use a lower resolution quadrupole TOF (Q-TOF)MS, as opposed to a high-resolution FT-ICR MS, usuallyemployed in top-down proteomic, further highlighting theaccessibility of the method. Their protein mixture containedBSA, ubiquitin (Ubi), cytochrome C (Cyt C), �-lactoglobulin(BLG), lysozyme (Lyso), and ribonuclease B (RNase B), andwas separated using traditional C18 RP column, with all theproteins eluting in less than 25 min. Reduction of disulfidebridges altered retention time (RT), however, the changes inRT were not proportional to the numbers and cannot serveas a discriminating criteria for disulfide-containing proteins.As expected, the authors confirm that RNase B and Lyso havefour disulfide bonds and BLG has two, whereas Ubi and CytC had none. More interesting, however, is the ability of Zhaoet al. to rapidly determine the number of disulfide bonds inBSA, an astounding 17, in two sequential runs of the reducedand nonreduced form of the protein, despite poor massresolution masking the isotopic distribution. Furthermore,confounding factors, such as PTMs (i.e. glycosylation) andprotein isoforms did not impede the proper identification ofdisulfide bond number.

It remains to be seen how far this method can be pushedin terms of sample complexity. Whole cell lysates, or evensubcellular fractions, present much more difficulty in termsof analysis than a simple mixture of six purified proteins.Systematic and unbiased comparison between the reducedand nonreduced sample runs will be needed to determinemolecular weight differences, especially in light of varyingRT. Additionally, the authors’ choice of using a LC-Q-TOF-MSapproach prevents prospective protein identification. Futurestudies using this method with MS/MS, and with higher res-olution instruments, will reveal unknown disulfide bond con-taining proteins. This would also allow investigation of howPTMs might affect disulfide formation, as major strength oftop-down proteomics. One other potential application of themethod is identifying quaternary structures between homod-imers linked by disulfide bridges. These have recently come tolight in redox-sensitive signaling proteins, such as NF�B [10]and Bak [11].

In summary, this novel method is easy to implement,provides crucial information about the number of disulfidebridges in a protein, and is likely to help future studies inter-rogating at disulfide bridges in cells and their regulation bythe cytosolic redox state.

Dr. Sadayappan was supported by National Institutes ofHealth grants R01 HL105826 and K02 HL114749.

The authors have declared no conflict of interest.

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