4 Results
4.1 Y2H screening as PPIs discovery tool
The Y2H-matrix screen approach is one of the most efficient methods to discover proteome-wide protein-protein interactions (PPIs) (Worseck et al., 2012). It produces high-quality, binary PPI data and is specifically suitable for detection of transient interactions. However, it provides a low sensitivity (Vinayagam et al., 2010; Yu et al., 2008). The sensitivity of individual Y2H systems is only 5 to 20 %, which is comparable to other PPI detection methods. There are several factors determining the sensitivity. For example each method detects its own subset of interactions which is called the “assay sensitivity”. Hence, sensitivity can be increased by combining different PPI detection methods (Sanderson, 2009; Stelzl and Wanker, 2006; Suter et al., 2008). Notably, this also holds true for different versions of the Y2H system. Provided that different Y2H systems are producing high precision data, parallel use of several Y2H setups will increase sensitivity which increases the workload (Figeys, 2008; Rajagopala et al., 2009; Schwartz et al., 2009). The “sampling sensitivity” is the fraction of all identifiable interactions that are found in a single trial of an assay performed. The sampling sensitivity in a Y2H-matrix screen is 45 % and approximately six screens are needed to reach 90 % saturation resulting in six times the workload (Venkatesan et al., 2009). Here, we describe a new workload reduced Y2H approach for comprehensive PPI mapping utilizing second generation sequencing.
4.1.1 Development of a new Y2H second generation sequencing screen
The overall aim is to reduce the workload and increase the sensitivity of the Y2H screen whilst producing high-quality PPI data. In Figure 6A and B the new Y2H second generation sequencing (Y2H-seq) screen is schematically presented in comparison to the standard Y2H-matrix screen, for one of four replicas. Ninety-six baits containing the proteins of interest were exemplarily tested for interactions against a set of preys here referred to as prey array. The prey array consisted of individually subcloned and characterized prey clones that expressed a particular prey protein in each well of a plate. In the Y2H-seq screen approach one prey array was pooled and subsequently split into four parts. Each of the parts was mixed with one of four pooled bait culture replicas. The mix was repeatedly spotted onto non-selective medium to allow mating. Hence, several thousand individual matings were performed. To select for interactions the mated yeast was transferred onto selective medium. Only diploid yeast containing a positive interaction pair of proteins can grow on selective medium. Plasmid DNA was isolated from grown yeast spots, prey plasmid inserts amplified and identified by second generation sequencing. Taking into account the number of obtained reads for each prey a ranked list of primary prey hits was generated.
Retest
identified preysidentification of primary interacting
preys via matrix
position
prey array selected baits
identification and confirmation of interacting proteins selected baits prey-bait interaction
prey array selected baits
second generation sequencing based identification and ranking of primary interacting preys
Y2H-matrix screen
Y2H-seq screen
38 MTP
700ml
10ml
152
/4MTP
38
/4MTP
1 MTP
Figure 6: Schematic representation of the Y2H-seq and Y2H-matrix screen
The new Y2H-seq screen (left) and the standard Y2H-matrix screen (right) are schematically compared for one of four replicas. Selected baits transformed with the protein of interested were screened against the prey array containing 14,268 prey clones. Baits were separately grown and then pooled. In the Y2H-seq approach preys were separately grown and then pooled, too. Small volume of highly concentrated pooled preys was mixed with highly concentrated pooled baits. The mixture of preys and baits was distributed into one microtiterplate (MTP). The MTP was stamped 36 times on non-selective medium. Hence, 13,824 individual matings were performed. After mating the diploid
yeast was transferred to selective medium. Diploid yeast containing interacting bait and prey can grow on selective medium. Plasmid DNA of the yeast colonies was isolated, prey inserts amplified, fragmented and sequenced in a Solexa HighSeq2000 sequencer. On the right hand the Y2H-matrix approach. Here, a large volume of pooled bait culture were needed to fill 38 MTP and mate every well separately with one of the 14,268 preys in the ordered array. After mating the diploid yeast was transferred to selective medium. Interacting preys and baits grow on selective medium. In the Y2H-matrix screen preys interacting with at least one bait of the pool were identified by their array position. The retest at the bottom is identical for both approaches. The identified preys were grown in flasks and separately mated against each replica of the baits. Interacting preys and baits grew on the selective medium.
In the Y2H-matrix screen every prey was tested separately against the pool of baits. Hence, every prey was mated with the pool of baits in a specific position in the prey array and the interacting preys were identified by their array position. The mating was performed in four replicas and therefore four ordered prey arrays were needed. By computational analysis a retest list containing primary prey hits was generated. In comparison to the Y2H-matrix workflow in the Y2H-seq approach the pool of preys was mated with the pool of baits, subsequently reducing the workload. Hence, the workload in the Y2H-seq approach is reduced.
The primary prey hits identified in the Y2H-matrix and in the Y2H-seq screen were verified in a retest approach (Figure 6C). In the retest preys were tested separately against selected baits. Thus, the interacting proteins were unambiguously identified. The retest layout was identical for both screens and hence resulted in the same high confidence PPIs.
In summary, the novel PPI Y2H-seq mating approach reduced the workload substantially in comparison to the standard Y2H-matrix approach whilst produces the same high confidence PPI data.