We report the coupling of a hybrid ionization source, matrix-assisted laser desorption electrospray ionization (MALDESI), to a Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS). The details of the source design and initial data are presented. Analysis of peptides and proteins ranging from 1 to 8.6 kDa resulted in high resolving power single-acquisition FT-ICR mass spectra with average charge-states highly correlated to those obtained by nanoESI, thus, providing strong evidence that the ESI process dictates the observed charge-state distribution. Importantly, unlike the recently introduced electrospray assisted laser desorption ionization (ELDI) source reported by Shiea and coworkers [1,2], the data we have obtained to date rely on the use of an organic acid matrix. The results presented herein provide insight into the charging mechanism of this emerging ionization approach, while also expanding the utility of FT-ICR MS for top-down protein and complex mixture analysis. T he field of mass spectrometry has grown tremendously over the past century due in large part to the continued development of more powerful ionization techniques, mass analyzer technologies, and ion dissociation techniques. New ionization techniques inherently increase the breadth of applications amenable to mass spectrometry, thereby driving the discovery, development, implementation, and refinement of mass analyzers and ion dissociation methods. The advent of electrospray ionization (ESI) [3] and matrixassisted laser desorption ionization (MALDI) [4,5] resulted in a paradigm shift in the biological sciences whereby intact biomacromolecules could be ionized and detected by mass spectrometry.The benefits of direct analysis of samples under ambient conditions with minimal or no sample pretreatment has led to the development of a wide range of novel ionization methods with potentially extraordinary impact in numerous fields (e.g., environmental, forensics, material science, biomedical). These include fused-droplet electrospray ionization (FD-ESI) [6], laser desorption atmospheric pressure chemical ionization (LD-APCI) [7], desorption electrospray ionization (DESI) [8], direct analysis in real time (DART) [9], atmospheric-pressure solids analysis probe (ASAP) [10], and electrospray-assisted laser desorption/ionization (ELDI) [1,2]. Interestingly, modern high-performance mass analyzers are largely based on hybrid technology (e.g., coupling of linear ion trap with a Fourier transform mass spectrometer [11]) and, similarly, so are these new ionization techniques.Electrospray ionization is inherently well-suited for FT-ICR MS due to the inverse relationship between cyclotron frequency and m/z [12]. Because cyclotron frequency dictates mass resolving power, mass accuracy, limits of detection, and top-down tandem MS efficiencies, the multiple charging (lower m/z) afforded by ESI provides significant advantages [13]. Matrixassisted laser desorption ionization (MALDI) can also be coupled to FT-ICR MS where it can be a very powerful tec...

During the past decade, the field of mass spectrometry imaging (MSI) has greatly evolved, to a point where it has now been fully integrated by most vendors as an optional or dedicated platform that can be purchased with their instruments. However, the technology is not mature and multiple research groups in both academia and industry are still very actively studying the fundamentals of imaging techniques, adapting the technology to new ionization sources, and developing new applications. As a result, there are an important variety of data file formats used to store mass spectrometry imaging data and concurrent to the development of MSi collaborative efforts have been undertaken to introduce common imaging data file formats. However, few free software packages to read and analyze files of these different formats are readily available. We introduce here MSiReader, a free open source application to read and analyze high resolution MSI data from the most common MSi data formats. The application is built on the Matlab platform (Mathworks, Natick, MA) and includes a large selection of data analysis tools and features. People who are unfamiliar with the Matlab language will have little difficult navigating the user friendly interface, and users with Matlab programming experience can adapt and customize MSiReader for their own needs.

A multi-omics quantitative integrative analysis of lignin biosynthesis can advance the strategic engineering of wood for timber, pulp, and biofuels. Lignin is polymerized from three monomers (monolignols) produced by a grid-like pathway. The pathway in wood formation of Populus trichocarpa has at least 21 genes, encoding enzymes that mediate 37 reactions on 24 metabolites, leading to lignin and affecting wood properties. We perturb these 21 pathway genes and integrate transcriptomic, proteomic, fluxomic and phenomic data from 221 lines selected from ~2000 transgenics (6-month-old). The integrative analysis estimates how changing expression of pathway gene or gene combination affects protein abundance, metabolic-flux, metabolite concentrations, and 25 wood traits, including lignin, tree-growth, density, strength, and saccharification. The analysis then predicts improvements in any of these 25 traits individually or in combinations, through engineering expression of specific monolignol genes. The analysis may lead to greater understanding of other pathways for improved growth and adaptation.

Protein cleavage-isotope dilution mass spectrometry (PC-IDMS) can be used to quantify proteins, with an isotope-labeled analogue of the peptide fragment used as an internal standard. Here, we investigate use of a standard LC-MS/MS platform for quantifying a model biomarker directly from serum by this technique. We synthesized a peptide (IVGGWECEK) identical to the N-terminal tryptic fragment of PSA but with each glycine containing two 13C atoms and one 15N atom. PSA-free human serum was denatured with urea followed by the introduction of PSA standard and the stable isotope labeled internal standard peptide. The sample was then proteolyzed with trypsin and subjected to quantification using LC-MS/ MS on a triple quadrupole mass spectrometer. A linear least squares calibration curve made from five different concentrations of PSA added to serum and digested (each made in triplicate and randomly injected three times) had a mean slope of 0.973 (SE = 0.023), intercept of -0.003 (SE = 0.022), and R2 of 0.971. Recovery of calibrators ranged from 70 to 85% with a mean run-to-run CV of 13% and a mean within-run CV of 5.7%. PC-IDMS is a promising technique for quantifying proteins covering a broad range of applications from standardizing immunoassays to monitoring post-translational modifications to quantifying newly discovered biomarkers prior to the development and implementation of an immunoassay, just to name a few. Issues surrounding the application of PC-IDMS for the absolute quantification of proteins include selection of a proteolytic fragment for quantification that can be cleaved and isolated reproducibly over a broad dynamic range, stable isotope labeled synthetic peptide standards that give consistent results, and LC-MS/MS methods that provide adequate sensitivity and reproducibility without creating impractical analysis times. The results presented here show that absolute quantification can be performed on the model biomarker PSA introduced into denatured serum when analyzed by LC-MS/MS. However, concerns still exist regarding sensitivity compared to existing immunoassays as well as the reproducibility of PC-IDMS performed in different matrixes.

The TripleTOF 5600 System, a hybrid triple quadrupole time-of-flight mass spectrometer, was evaluated to explore the key figures of merit in generating peptide and protein identifications which included spectral acquisition rates, data quality, proteome coverage, and biological depth. Employing a Saccharomyces cerevisiae tryptic digest, careful consideration of several performance features demonstrated that the speed of the TripleTOF contributed most to the resultant data. The TripleTOF system was operated with 8, 20, and 50 MS/MS events in an effort to compare to other MS technologies and to demonstrate the abilities of the instrument platform.