Toward chromatographic analysis of interacting protein networks

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Abstract

Protein complexes, collectively referred to as the cellular interactome, appear to play a major role in cellular regulation. At present it is thought that the interactome could be composed of hundreds of protein assemblies. The objective of the work described here was to examine the prospect that chromatographic methods widely used in the preparative isolation of native proteins could be incorporated into global proteomics methods in such a way that the primary structure of protein complexes of sufficient stability to survive chromatography could be recognized along with their participation in protein complexes. Because wide differences in sizes are a unique feature of protein complexes, size-exclusion chromatography (SEC) was incorporated into all the fractionation strategies examined. Anion-exchange chromatography (AEC) and hydrophobic-interaction chromatography (HIC) were also examined because of the broad utility that these methods have shown in the preparation of proteins with native structure. Slightly more than a third of all proteins identified in yeast lysates were found to elute from SEC, AEC, and HIC columns with an apparent molecular weight much higher than that predicted from their parent gene. These results were interpreted to mean that these proteins were migrating through columns as components of protein complexes. Based on studies with multidimensional SEC  RPLC (reversed-phase liquid chromatography), AEC  SEC, and HIC  SEC systems, it was concluded that recognition of proteins in complexes could be easily incorporated into multidimensional chromatographic methods for global proteomics when at least one of the fractionation dimensions included SEC of native proteins.

Introduction

Evidence is accumulating that many proteins reside in multi-subunit complexes ranging in size from a few proteins to large assemblies of 50 or more subunits [1]. In fact, protein–protein interactions are fundamental to a number of biological processes [2]. The sum of all these interactions is referred to as either the cellular protein interaction network or simply the ‘interactome’. Given the importance of these complexes, there is great interest in recognizing them in cellular extracts, determining their composition, studying the dynamics of their formation and dissociation, and establishing the role they play in certain diseases.

Although liquid chromatography has been widely used in protein structure analysis, it is yet to be determined whether chromatographic systems will be of equally broad utility in the study of protein interaction networks. A major question is the degree to which protein complexes will survive adsorption and desorption from column surfaces along with the requisite changes in mobile-phase composition and dilution inherent in liquid chromatography. Small numbers of protein complexes have been isolated in a variety of ways ranging from affinity [3], [4] and ion-exchange chromatography [5] to immobilized metal affinity chromatography (IMAC) [6]. It has been shown that a protein complex is more likely than a small protein to desorb from a chromatographic sorbent of low stationary-phase density than the higher one because it covers a larger surface area when adsorbed and can still make contact with multiple, widely distributed stationary-phase groups [7]. Adsorption of proteins to surfaces generally involves a small number of sites as seen with lactate dehydrogenase [8]. This fact and stationary-phase dilution has been exploited to create sorbent surfaces in which stationary-phase groups are sterically complimentary to individual groups on the surface of a specific protein [9]. From this it is seen that diluted stationary phases can become very stereoselective. It is doubtful that the stationary-phase dilution technique can be broadly applied. Moreover, it has been shown that some of the tetrameric isoforms of lactate dehydrogenase are weakly adsorbed to an anion exchanger, while others are strongly adsorbed. Within the isoforms of a single protein complex, this approach would fail.

Single, targeted protein complexes have also been isolated via tandem affinity chromatography (TAP) [3], [4] by adding affinity selectable polypeptide tags at either the amino- or carboxy-termini of two proteins that are putatively in the complex being targeted. These tagged proteins are then sequentially selected in two affinity chromatography steps [10], bringing with them other members of the protein complex. Tags are designed to interact with an affinity column weakly so that the complex can be eluted without dissociation of other members in the complex. Unfortunately, identifications with this method are not very reproducible. It remains to be determined whether this problem arises from the chromatography or failure to control some other experimental variable that causes dissociation of members from the complex. The fact that genetic engineering is necessary to target a complex is another complication. This means that each complex must be studied individually and throughput will be low. Moreover, global searches for complexes that change in association with specific stimuli will be difficult and the method will be of limited utility in the study of human subjects where genetic manipulation is not possible.

Antibody pull-down methods are another approach to studying protein–protein association. Co-immunoaffinity purification of proteins in a complex is fairly gentle and reproducible but suffers from a different set of problems than the TAP method. A protein can be part of multiple complexes, as in the case of so-called “hub proteins”. Hub proteins associate with multiple protein complexes. Antibodies targeting any protein that resides in multiple complexes will pull them all down together, making them appear to be part of a single large complex. The antibody pull-down approach also suffers from the problem that the epitope can be in the interface between the protein and the rest of the complex. When the protein antigen binds to the antibody, in this case it is precluded from association with the complex.

The yeast-two hybrid (YTH) [11], [12], [13] method provides still another way to recognize protein–protein interactions through tagged proteins, but does so in vivo and only recognizes binary interactions. The requisite polypeptide tags in the YTH approach are often the DNA-binding and activation domains of a transcription factor, each fused to one of the members of a potential interacting protein pair. Interaction of the two fusion proteins through putative members of the protein complex in vivo causes the formation of a functional transcription factor that in turn links transcription of some reporter gene(s) to a change in the cell phenotype. Advantages of the YTH method are that the protein–protein interaction is being examined in vivo and even weak association of proteins can be detected. Limitations of the method are that it gives false positives and it is not possible to examine all the members of a protein complex simultaneously. This diminishes the utility of the method for examining the dynamics [14] of complex formation as a function of various stimuli [15]. There is also poor overlap between observations made with the TAP and YTH methods.

A system is needed that allows multiple protein complexes to be examined simultaneously along with comparing and validating results from the TAP, YTH, and antibody pull-down methods. This paper focuses on the potential of chromatography systems to do that. It has been shown above that protein complexes remain partially or totally intact during mild forms of elution from affinity chromatography columns. The work described here explores the extent to which protein interaction partners from a yeast lysate remain associated during other modes of chromatography, such as size exclusion, ion-exchange, and hydrophobic interaction chromatography. The advantage of these methods is that they allow many protein complexes to be recognized and studied at the same time without genetic manipulation. When these higher resolution, multidimensional methods were applied to yeast lysates, many proteins were shown to remain in complexes as indicated by their co-migration through at least two or more orthogonal dimensions of chromatography. These co-migrating proteins were frequently found to have been identified as interaction partners by the YTH or TAP methods as well. It was found that co-migration through multiple dimensions of chromatography can be the basis for identifying members of a complex without actually isolating the complex.

Section snippets

Materials

Potassium phosphate monobasic, d-glucose, and HPLC grade acetonitrile were purchased from Mallinckrodt Chemicals (Phillipsburg, NJ, USA). 4-Cyano-hydroxy-cinnamic acid (CHCA), vitamin B12, cytidine, N-p-tosyl-phenylalanine chloromethyl ketone (TPCK)-treated trypsin, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), iodoacetic amide (IAA), N-acetyl cysteine, trifluoroacetic acid (TFA), vitamin B12, cytidine, and a matrix-assisted laser desorption ionization–time of flight–time of

Results

A goal of the work described here was to evaluate whether protein interaction partners identified by the YTH or TAP methods remain together during passage through a variety of chromatographic modes ranging from size exclusion and ion-exchange chromatography to hydrophobic interaction chromatography. The yeast proteome was used as a source of protein complexes because of the large amount of yeast two-hybrid and tandem affinity purification data available showing that protein complexes exist in

Discussion

Identification of protein interaction partners is plagued by a lack of correlation between the various methods and poor within-method reproducibility [3], [4]. The value of SEC in this context is that results from the YTH, antibody pull-down, or TAP methods can be validated by showing that proteins identified by these methods are indeed found in cell lysates at higher apparent molecular weight than predicted by genomics. Moreover, proteomic analysis of SEC fractions allows it to be determined

Conclusion

It is concluded from this study of the protein complexes in a S. cerevisiae lysate, many remain intact during SEC in addition to AEC and HIC. It is further concluded that while in a complex, all the component proteins migrate as a single entity and that protein complexes have unique chromatographic behavior different from that of the individual components in a complex. The strongest indicator of residence in a complex is that the apparent molecular weight of a protein is much higher than that

Acknowledgements

The authors gratefully acknowledge the support of this work by multiple organizations and individuals. The National Institutes of Health has provided financial support for this work through an Ageing Grant (1P30- AG13319). Beckman Coulter kindly provided the ProteomeLab PF 2D chromatographic separation system for all the multidimensional protein fractionation along with technical support. The laboratories of Professors Phil Low and Christina Hrycyna in the Chemistry Department at Purdue allowed

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