The ability of mass spectrometry to generate intact biomolecular ions efficiently in the gas phase has led to its widespread application in metabolomics, proteomics, biological imaging, biomarker discovery and clinical assays (namely neonatal screens). Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization have been at the forefront of these developments. However, matrix application complicates the use of MALDI for cellular, tissue, biofluid and microarray analysis and can limit the spatial resolution because of the matrix crystal size (typically more than 10 mum), sensitivity and detection of small compounds (less than 500 Da). Secondary-ion mass spectrometry has extremely high lateral resolution (100 nm) and has found biological applications although the energetic desorption/ionization is a limitation owing to molecular fragmentation. Here we introduce nanostructure-initiator mass spectrometry (NIMS), a tool for spatially defined mass analysis. NIMS uses 'initiator' molecules trapped in nanostructured surfaces or 'clathrates' to release and ionize intact molecules adsorbed on the surface. This surface responds to both ion and laser irradiation. The lateral resolution (ion-NIMS about 150 nm), sensitivity, matrix-free and reduced fragmentation of NIMS allows direct characterization of peptide microarrays, direct mass analysis of single cells, tissue imaging, and direct characterization of blood and urine.

Metal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability. Consequently, metalloproteins have key roles in most biological processes, including respiration (iron and copper), photosynthesis (manganese) and drug metabolism (iron). Yet, predicting from genome sequence the numbers and types of metal an organism assimilates from its environment or uses in its metalloproteome is currently impossible because metal coordination sites are diverse and poorly recognized. We present here a robust, metal-based approach to determine all metals an organism assimilates and identify its metalloproteins on a genome-wide scale. This shifts the focus from classical protein-based purification to metal-based identification and purification by liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) to characterize cytoplasmic metalloproteins from an exemplary microorganism (Pyrococcus furiosus). Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used (cobalt, iron, nickel, tungsten and zinc; 83 peaks) plus metals the organism was not thought to assimilate (lead, manganese, molybdenum, uranium and vanadium; 75 peaks). Purification of eight of 158 unexpected metal peaks yielded four novel nickel- and molybdenum-containing proteins, whereas four purified proteins contained sub-stoichiometric amounts of misincorporated lead and uranium. Analyses of two additional microorganisms (Escherichia coli and Sulfolobus solfataricus) revealed species-specific assimilation of yet more unexpected metals. Metalloproteomes are therefore much more extensive and diverse than previously recognized, and promise to provide key insights for cell biology, microbial growth and toxicity mechanisms.

Silylation chemistry on porous silicon provides for ultrahigh sensitivity and analyte specificity with desorption/ionization on silicon mass spectrometry (DIOS-MS) analysis. Here, we report that the silylation of oxidized porous silicon offers a DIOS platform that is resistant to air oxidation and acid/base hydrolysis. Furthermore, surface modification with appropriate hydrophobic silanes allows analytes to absorb to the surface via hydrophobic interactions for direct analyte extraction from complex matrixes containing salts and other nonvolatile interferences present in the sample matrix. This enables rapid cleanup by simply spotting the sample onto the modified DIOS target and removing the liquid phase containing the interferences. This approach is demonstrated in the analysis of protein digests and metabolites in biofluids, as well as for the characterizing of inhibitors from their enzyme complex. An unprecedented detection limit of 480 molecules (800 ymol) for des-Arg(9)-bradykinin is reported on a pentafluorophenyl-functionalized DIOS chip.

Dense arrays of single-crystal silicon nanowires (SiNWs) have been used as a platform for laser desorption/ionization mass spectrometry of small molecules, peptides, protein digests, and endogenous and xenobiotic metabolites in biofluids. Sensitivity down to the attomole level has been achieved on the nanowire surfaces by optimizing laser energy, surface chemistry, nanowire diameter, length, and growth orientation. An interesting feature of the nanowire surface is that it requires lower laser energy as compared to porous silicon and MALDI to desorb/ionize small molecules, therefore reducing background ion interference. Taking advantage of their high surface area and fluid wicking capabilities, SiNWs were used to perform chromatographic separation followed by mass analysis of the separated molecules providing a unique platform that can integrate separation and mass spectrometric detection on a single surface.

A new and general methodology is described for the targeted enrichment and subsequent direct mass spectrometric characterization of sample subsets bearing various chemical functionalities from highly complex mixtures of biological origin. Specifically, sample components containing a chemical moiety of interest are first selectively labeled with perfluoroalkyl groups, and the entire sample is then applied to a perfluoroalkyl-silylated porous silicon (pSi) surface. Due to the unique hydrophobic and lipophobic nature of the perfluorinated tags, unlabeled sample components are readily removed using simple surface washes, and the enriched sample fraction can then directly be analyzed by desorption/ionization on silicon mass spectrometry (DIOS-MS). Importantly, this fluorous-based enrichment methodology provides a single platform that is equally applicable to both peptide as well as small molecule focused applications. The utility of this technique is demonstrated by the enrichment and mass spectrometric analysis of both various peptide subsets from protein digests as well as amino acids from serum.

Nanostructure-initiator mass spectrometry (NIMS) is a highly sensitive, matrix-free technique that is well suited for biofluid analysis and imaging of biological tissues. Here we provide a new technical variation of NIMS to analyze carbohydrates and steroids, molecules that are challenging to detect with traditional mass spectrometric approaches. Analysis of carbohydrates and steroids was accomplished by spray depositing NaCl or AgNO3 on the NIMS porous silicon surface to provide a uniform environment rich with cationization agents prior to desporption of the fluorinated polymer initiator. Laser desorption/ionization of the ion-coated NIMS surface allowed for Na+ cationization of carbohydrates and Ag+ cationization of steroids. The reliability of the approach is quantitatively demonstrated with a calibration curve over the physiological range of glucose and cholesterol concentrations in human serum (1 – 200 μM). Additionally we illustrate the sensitivity of the method by showing its ability to detect carbohydrates and steroids down to the 800-amol and 100-fmol levels, respectively. The technique developed is well suited for tissue imaging of biologically significant metabolites such as sucrose and cholesterol. To highlight its applicability, we used cation-enhanced NIMS to image the distribution of sucrose in a Gerbera jamesonii flower stem and the distribution of cholesterol in a mouse brain. The flower stem and brain sections were placed directly on the ion-coated NIMS surface without further preparation and analyzed directly. The overall results reported underscore the potential of NIMS to analyze and image chemically diverse compounds that have been traditionally challenging to observe with mass spectrometry-based techniques.

A surface-based laser desorption/ionization mass spectrometry assay that makes use of Desorption/Ionization on Silicon Mass Spectrometry (DIOS-MS) has been developed to monitor enzyme activity and enzyme inhibition. DIOS-MS has been used to characterize inhibitors from a library and then to monitor their activity against selected enzyme targets, including proteases, glycotransferase, and acetylcholinesterase. An automated DIOS-MS system was also used as a high-throughput screen for the activity of novel enzymes and enzyme inhibitors. On two different commercially available instruments, a sampling rate of up to 38 inhibitors per minute was accomplished, with thousands of inhibitors being monitored. The ease of applying mass spectrometry toward developing enzyme assays and the speed of surface-based assays such as DIOS for monitoring inhibitor effectiveness and enzyme activity makes it attractive for a broad range of screening applications.

Perfluorinated surfactants are demonstrated to dramatically enhance desorption/ionization on fluorinated silicon (DIOS) mass spectrometry. Perfluorooctanesulfonic acid improved the signal-to-noise ratio of tryptic digests and gave a 3-fold increase in the number of peptides identified. Similar results were also obtained using perfluoroundecanoic acid; yet among the seven different surfactants tested, controls such as nonfluorinated sodium dodecyl sulfate or fluorinated molecules with minimal surfactant activity did not enhance the signal. The same surfactants also enhanced the DIOS-MS signal of amino acids, carbohydrates, and other small organic compounds. The signal enhancement may be facilitated by the high surface activity of the perfluorinated surfactants on the fluorinated silicon surfaces allowing for a higher concentration of analyte to be absorbed.