In the search for alternative inert surfaces replacing silicon chips in Desorption/Ionization On porous Silicon (DIOS)-like mass spectrometry analyses, nanostructured silicon-based NALDI chips were evaluated in Laser Desorption/Ionization (LDI) of peptides. Comparisons were made using commercially available DIOS chips (MassPREP-DIOS-target), amorphous carbon powder from lead pencil and porous silica gel used for chromatographic purposes as reference supports. A set of synthetic model peptides presenting variable amino acid sequences of various lengths was analyzed under all conditions. The LDI responses of the four 'matrix-free' techniques were compared, especially in terms of peptide detection sensitivity and overall experiment robustness.

We report our contribution to the systematic investigation of peptide fragmentations performed on high-performance Tof equipment, operating in MS and MS/MS modes, such as ESI-QqTof and MALDI-Tof/Tof instruments that are commonly available today in proteomic laboratories. Whereas the former analyzer's configuration provides low-energy collision-induced dissociations (CID), the latter allows tunable activation methods of the selected parent ion to induce either metastable laser-induced dissociations (LID) or high-energy CID ('gas on spectra LID'). Fragmentation of the monoprotonated ion of 53 peptides (FW 807-2853 g/mol) was undertaken upon low-energy CID on an ESI-QTof mass spectrometer (Waters) as well as high-energy CID and LID conditions on a MALDI Ultraflex mass spectrometer (Bruker). Systematic comparison of MS/MS spectra provided useful information on the performance of each piece of equipment for efficient peptide sequencing and also insights into the observed fragmentation behaviors.

We investigated a variant of desorption/ionization on porous silicon (DIOS) mass spectrometry utilizing an aqueous suspension of either porous silica gel or porous alumina (pore size of 60 and 90 Å, respectively). Laser desorption/ionization (LDI) from samples directly deposited on a stainless steel surface without any inorganic substrates was also achieved. Synthetic peptides designed to cover large sequence diversity constituted our model compounds. Sample preparation, including material conditioning, peptide solubilization, and deposition protocol onto standard matrix-assisted laser desorption/ionization (MALDI) probe, as well as ionization source tuning were optimized to perform sensitive reproducible LDI analyses. The addition of either a cationizing agent or an alkali metal scavenger to the sample suspension allowed modification of the ionization output. Comparing hydrophilic silica gel to hydrophobic reversed-phase silica gel as well as increasing material pore size provided further insights into desorption/ionization processes. Furthermore, mixtures of peptides were analyzed to probe the spectral suppression phenomenon when no interfering organic matrix was present. The results gathered from synthetic peptide cocktails indicated that LDI mass spectrometry on silica gel or alumina constitutes a promising complementary method to MALDI in proteomics for peptide mass fingerprinting. [19]. These LDI methods, sometimes referred to as "matrix-free", produce mass spectra that exhibit less matrix ions in the low mass range compared with the chemical noise generated by the organic matrices used in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Such chemical noise that usually pollutes the low mass range of MALDI mass spectra and that may complicate the detection of small molecules is less pronounced in the DIOS mass spectra.Nevertheless, the drawback of such an approach is in the preparation of the silicon material. To present adequate physical properties [20], silicon surfaces are conditioned via a galvanostatic etching procedure from Si wafer to produce pores with nanometer diameters. Commercially available ready-to-use DIOS-chips can also be mounted on modified MALDI plates [20]. Otherwise, silicon powders (5 to 50 nm particle size) were investigated as a substitute to DIOS chips to make the overall sample preparation simpler and safer [18]. Provided that the appropriate powder preparation was performed (including particle etching by HF, oxidation by HNO 3 , and derivatization of the generated SiOH groups), such silicon nanopowder method was found to exhibit the same characteristics as DIOS mass spectrometry.Porous silicon is a material that presents (1) a large active surface and (2) a strong absorbance in the UV range. Such a form of silicon was thus found particularly suitable for LDI mass spectrometry with irradiation at 337 nm, the pore size and the surface chemical nature being the most important parameters as stated by Siuzdak and coworkers in their early publications on DIOS [20...