MALDI In-Source Photooxidation Reactions for Online Peptide Tagging This work is supported by NSFC (20775016, 20575013, 20525519), 973 Program (2007CB714506), Shanghai Leading Academic Discipline B109, and the Swiss-China cooperation research program

As one of the most widely used ionization techniques, MALDI or other related techniques such as surface-enhanced laser desorption ionization can transfer neutral solid-state samples to gas-phase ions for study in proteomics. Even if the underlying physicochemical phenomena occurring in these processes are still a matter of debate, the applications have been widely used. Various substrates and matrices have been employed to endow additional functions on the target plates either to assist the ion yield for nonvolatile analytes or to bring about specific interactions with target biomolecules from complex samples. Herein, we propose a new concept for driving in-source photoelectrochemical redox reactions under UV laser irradiation and designing a highly porous TiO2 photoelectrode on a steel plate to achieve very efficient electron-transfer reactions with electron donors or acceptors present in the sample or added to the sample. The photoelectrode is made of TiO2 nanoparticles, similar to that used for the design of dye-sensitized solar cells. Under UV irradiation, the large number of TiO2 nanoparticles absorb photons generating electron–hole pairs and therefore acting as photosensitizers to drive very efficiently electron-transfer reactions. For MALDI-MS analysis, the photoelectrode is coated with the sample and a matrix overlayer (a-cyano-4-hydroxycinnamic acid; CHCA) is added to assist light-energy absorption and protonation of the sample. One advantage of the photoelectrode-modified plate is that it enables the determination of the oxidation or reduction products of a given molecule. If this oxidation/ reduction induces fragmentation of the analytes, the photoelectrode generates in-source decay upon irradiation. Alternatively, the oxidation/reduction products may in turn further react with other molecules, and all the products of these electron-transfer chain reactions can be directly studied by mass spectrometry. To illustrate this principle, we use the addition reaction of oxidized hydroquinone on cysteine-containing peptides, which has previously been demonstrated with electrospray ionization. Indeed, selective tagging of specific amino acids is an interesting protocol in protein profiling by peptide mass fingerprinting. We show here that a selective and efficient in-source tagging reaction can be developed by combining photoelectrochemical reactions with sample ionization for high-throughput analysis with MALDI-MS. In this case, the unique peptides can be revealed from the digested proteomes. Such a photoinitiated tagging strategy is simple and does not cause sample loss, thus providing powerful information to facilitate accurate and comprehensive protein identification. For this purpose, a photoelectrode array based on TiO2 nanoparticles was fabricated both as a photoreactive substrate and as a porous support to deposit the sample and the matrix on a stainless steel target plate for MALDI analysis (see the Supporting Information). This mesoporous photoelectrode exhibiting a large specific surface area enables a high oxidation capacity, as the photon capture cross-section is very large. Figure 1 shows an illustration of the tagging