A new approach to nano scale protein analysis using chemical inkjet printing

The ChIP-1000 chemical inkjet printing technology offers researchers a new approach for automatic protein processing, identification and characterisation. This unique technology platform was designed for executing micro-scale on-membrane chemistry which will have widespread applications in biomedical research and biomarker discovery.
Many proteins of interest in multiple proteomics investigations are available only in limited amounts and unlike DNA, there is no process equivalent to PCR for amplification of proteins. Using current methods, an entire sample on a protein array (e.g. 1D or 2D gel) is often sacrificed for identification and perhaps a partial characterisation. In particular the option of performing different analyses on a single target without having to repeat the whole separation procedure would be helpful for samples limited in amount or extracted by time-consuming preparation steps. The option to archive these samples and use them at a later stage is also an important issue. The new approach using chemical inkjet printing technology should help to address at least some of these challenges.

The ChIP-1000 (Figure 1) uses four piezoelectric devices for micro-dispensing chemical or biochemical solvents in the nano litre range to specific target locations (Figure 2). A common method is to immobilize proteins after 1D or 2D electrophoretic separation on a polymer membrane. Under these conditions samples can be archived easily for several months. The instrument offers two stages in a conventional 96 well plate format to mount on-membrane samples. In these areas each printing device can precisely deliver pico-litre droplets of reagent so that the solvent finally covers an area of 100–400 µm in diameter. For instance, a 2D gel spot can therefore be used for different analyses since one experiment just needs a fraction of a spot (Figure 3). As the instrument can focus on specific surface targets, it opens also new horizons in working with critical biological samples (e.g. brain tissue).
A large variety of established protocols are available which can be used for protein identification and characterisation with and without different derivation steps. Some of these protocols are designed for MS detection - others can be used for antigen discovery, or so-called nanowestern blotting.
Characterisation methods
For direct identification of proteins a stained membrane is fixed to a
MALDI target by conductive tape. The software allows the direct selection of areas of interest (e.g. protein bands or spots) after an image of the membrane is obtained by the scanning device imbedded into the ChIP-1000. Depending on the chosen method, each area can be separated into multiple print positions on which different enzymatic or chemical reactions are applied. Unlike time-consuming in-gel digestion with peptide extraction and C18 ZipTip clean up steps, the ChIP approach can save time and resources. For PMF analysis, the first printing is carried out with a specific solution to pretreat the position allowing the enzyme (e.g. trypsin, GluC) access to the membrane and digestion of the protein of interest. After a three hour digest the matrix is finally applied to the same print position. During this printing step the salt from the digestion buffer is also removed from the membrane. A volume of each solvent between 20 and 100 nanolitre is generally printed after adjusting and optimising of the droplet. The number of iteration cycles, in which the total volume is finally applied can be defined, which also influences the diameter of the reaction area (Figure 3). For protein identification the print coordinates are exported to the MALDI mass spec instrument so that the peptides are ionised directly from the membrane. A comparison of a tryptic in-gel and an on-membrane digest of different E. coli proteins with a molecular weight between 10 and 80 kDa showed on average a slightly higher sequence coverage for the in-gel digestion. However, with the ChIP approach some remaining sample was used for a second enzymatic digestion, which then further increased the sequence coverage.
The analysis of glycans is another established method, which can be performed in a miniaturized fashion. Peptidoglycans are digested by an on-membrane cleavage with Endoglycosidase PNGaseF to release attached N-linked glycans. The procedure is similar to the PMF analysis, but for glycans MALDI ionisation is less sensitive than electrospray ionisation, so the released glycans are generally aspirated from the print position and analysed via ESI-MS. The ChIP approach can also be used for a sequential deglycosylation with different exoglycosidases to obtain more structural information from the glycans.
The detection of antigens can be performed with the nanowestern approach. Again, the ChIP shows distinct advantages as it consumes less antibody solution and the preparation time is reduced in comparison with the conventional western blot.
Figure 5 shows an example of a ChIP nanowestern blot analysis versus a conventional method. This experiment focussed on the investigation of the serine and threonine phosphorylation of nonmuscle myosin heavy chain protein in mast cells during the antigen-induced secretory process. Four different activation periods were chosen, the lysed samples were separated using a 1D SDS gel and blotted on a PVDF membrane. For the conventional incubation with three different antibodies (anti-nonmuscle-myosin, anti-phospho-serine and anti-phospho-threonine) an individual gel for each assay was produced, whereas for the ChIP approach each antibody was used on the same band.
Conclusion
In comparison the results obtained with the ChIP are very similar to those obtained using conventional western blotting methods. However, the use of the inkjet technology for antibody binding studies is much faster, requires fewer gels or blots and uses significantly less antibody when compared with conventional western strategies. The flexibility of the ChIP speaks for itself and is sure to attract major interest as scientists adapt their own protocols for use with the ChIP approach in order to generate more information while saving sample material and time.
The ChIP 1000 has been chosen as one of the 100 most technologically significant products introduced to the market last year. Awarded with the R&D 100 award 2004 of R&D Magazine, highlighting a very competitive year of nominations. Jointly developed by Shimadzu Biotech and Proteome Systems.