James I. Langridge

The detection, correlation, and comparison of peptide precursor and product ions from data independent LC-MS with data dependant LC-MS/MS.

The detection, correlation, and comparison of peptide and product ions from a data independent LC‐MS acquisition strategy with data dependent LC‐MS/MS is described. The data independent mode of acquisition differs from an LC‐MS/MS data acquisition since no ion transmission window is applied with the first mass analyzer prior to collision induced disassociation. Alternating the energy applied to the collision cell, between low and elevated energy, on a scan‐to‐scan basis, provides accurate mass precursor and associated product ion spectra from every ion above the LOD of the mass spectrometer. The method therefore provides a near 100% duty cycle, with an inherent increase in signal intensity due to the fact that both precursor and product ion data are collected on all isotopes of every charge‐state across the entire chromatographic peak width. The correlation of product to precursor ions, after deconvolution, is achieved by using reconstructed retention time apices and chromatographic peak shapes. Presented are the results from the comparison of a simple four protein mixture, in the presence and absence of an enzymatically digested protein extract from Escherichia coli. The samples were run in triplicate by both data dependant analysis (DDA) LC‐MS/MS and data‐independent, alternate scanning LC‐MS. The detection and identification of precursor and product ions from the combined DDA search results of the four protein mixture were used for comparison to all other data. Each individual set of data‐independent LC‐MS data provides a more comprehensive set of detected ions than the combined peptide identifications from the DDA LC‐MS/MS experiments. In the presence of the complex E. coli background, over 90% of the monoisotopic masses from the combined LC‐MS/MS identifications were detected at the appropriate retention time. Moreover, the fragmentation pattern and number of associated elevated energy product ions in each replicate experiment was found to be very similar to the DDA identifications. In the case of the corresponding individual DDA LC‐MS/MS experiment, 43% of the possible detectable peptides of interest were identified. The presented data illustrates that the time‐aligned data from data‐independent alternate scanning LC‐MS experiments is highly comparable to the data obtained via DDA. The obtained information can therefore be effectively and correctly deconvolved to correlate product ions with parent precursor ions. The ability to generate precursor‐product ion tables from this information and subsequently identify the correct parent precursor peptide will be illustrated in a companion manuscript.

A novel precursor ion discovery method on a hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer for studying protein phosphorylation

A tandem quadrupole time-of-flight (Q-TOF) mass spectrometer has been programmed such that phosphorylated peptides can automatically be discovered and identified in a way similar to that of the use of precursor ion or neutral loss scanning, but without the need to scan the quadrupole mass filter. Instead, the method capitalizes on the innate capability of the Q-TOF to record mass spectra and product ion spectra quickly, with good sensitivity and with good mass accuracy. Alternate mass spectra, with and without fragmentation, are recorded at high and low collision energy with the quadrupole operating in wideband mode. The method of analysis is both compatible with and dependant on liquid chromatography for separation of complex mixtures. The method has been demonstrated by searching for the neutral loss of 98 Da (H3PO4) from phosphoserine and phosphothreonine residues, or for the phosphorylated immonium ion at m/z 216 from phosphotyrosine. The method also incorporates acquisition of the product ion spectrum from any candidate precursor ions, thereby allowing confirmation of the neutral loss or product ion and providing additional sequence information to assist identification of the protein and assign the site of phosphorylation.

Ion mobility derived collision cross sections to support metabolomics applications.

Metabolomics is a rapidly evolving analytical approach in life and health sciences. The structural elucidation of the metabolites of interest remains a major analytical challenge in the metabolomics workflow. Here, we investigate the use of ion mobility as a tool to aid metabolite identification. Ion mobility allows for the measurement of the rotationally averaged collision cross-section (CCS), which gives information about the ionic shape of a molecule in the gas phase. We measured the CCSs of 125 common metabolites using traveling-wave ion mobility-mass spectrometry (TW-IM-MS). CCS measurements were highly reproducible on instruments located in three independent laboratories (RSD < 5% for 99%). We also determined the reproducibility of CCS measurements in various biological matrixes including urine, plasma, platelets, and red blood cells using ultra performance liquid chromatography (UPLC) coupled with TW-IM-MS. The mean RSD was < 2% for 97% of the CCS values, compared to 80% of retention times. Finally, as proof of concept, we used UPLC–TW-IM-MS to compare the cellular metabolome of epithelial and mesenchymal cells, an in vitro model used to study cancer development. Experimentally determined and computationally derived CCS values were used as orthogonal analytical parameters in combination with retention time and accurate mass information to confirm the identity of key metabolites potentially involved in cancer. Thus, our results indicate that adding CCS data to searchable databases and to routine metabolomics workflows will increase the identification confidence compared to traditional analytical approaches.

Analysis and Quantification of Diagnostic Serum Markers and Protein Signatures for Gaucher Disease

Novel approaches for the qualitative and quantitative proteomics analysis by nanoscale LC-MS applied to the study of protein expression response in depleted and undepleted serum of Gaucher patients undergoing enzyme replacement therapy are presented. Particular emphasis is given to the method reproducibility of these LC-MS experiments without the use of isotopic labels. The level of chitotriosidase, an established Gaucher biomarker, was assessed by means of an absolute concentration determination technique for alternate scanning LC-MS generated data. Disease associated proteins, including fibrinogens, complement cascade proteins, and members of the high density lipoprotein serum content, were recognized by various clustering methods and sorting and intensity profile grouping of identified peptides. Condition-unique LC-MS protein signatures could be generated utilizing the measured serum protein concentrations and are presented for all investigated conditions. The clustering results of the study were also used as input for gene ontology searches to determine the correlation between the molecular functions of the identified peptides and proteins.

Ion Mobility-Derived Collision Cross Section As an Additional Measure for Lipid Fingerprinting and Identification

Despite recent advances in analytical and computational chemistry, lipid identification remains a significant challenge in lipidomics. Ion-mobility spectrometry provides an accurate measure of the molecules’ rotationally averaged collision cross-section (CCS) in the gas phase and is thus related to ionic shape. Here, we investigate the use of CCS as a highly specific molecular descriptor for identifying lipids in biological samples. Using traveling wave ion mobility mass spectrometry (MS), we measured the CCS values of over 200 lipids within multiple chemical classes. CCS values derived from ion mobility were not affected by instrument settings or chromatographic conditions, and they were highly reproducible on instruments located in independent laboratories (interlaboratory RSD < 3% for 98% of molecules). CCS values were used as additional molecular descriptors to identify brain lipids using a variety of traditional lipidomic approaches. The addition of CCS improved the reproducibility of analysis in a liquid chromatography-MS workflow and maximized the separation of isobaric species and the signal-to-noise ratio in direct-MS analyses (e.g., “shotgun” lipidomics and MS imaging). These results indicate that adding CCS to databases and lipidomics workflows increases the specificity and selectivity of analysis, thus improving the confidence in lipid identification compared to traditional analytical approaches. The CCS/accurate-mass database described here is made publicly available.

Polycomb Group Proteins as Epigenetic Mediators of Neuroprotection in Ischemic Tolerance

Polycomb group proteins protect neurons from injury through a mechanism involving decreased potassium channel function. Promoting Tolerance A temporary or permanent interruption of blood flow (ischemia) to the brain can lead to brain injury, with potentially devastating consequences, such as stroke. Intriguingly, previous exposure to a brief period of ischemia that is not itself sufficient to cause brain injury can protect against a later, more prolonged period of ischemia. This phenomenon, called ischemic tolerance, is associated with a general suppression of gene expression in the tolerant brain. Paradoxically, the tolerant state depends on protein synthesis. Stapels et al. performed proteomic analyses of mouse brains subjected to various ischemic conditions and found, in ischemic-tolerant brains, an increase in the abundance of polycomb group proteins, which act as transcriptional repressors. Further analyses of both mouse brain and cultured cells indicated that the polycomb proteins SCMH1 and BMI1 inhibited potassium channel abundance and activity and that this represented a mechanism underlying ischemic tolerance. A further understanding of the mechanisms underlying tolerance could open the door to new therapies for ischemic stroke. Exposing the brain to sublethal ischemia affects the response to a subsequent, otherwise injurious ischemia, resulting in transcriptional suppression and neuroprotection, a response called ischemic tolerance. Here, we show that the proteomic signature of the ischemic-tolerant brain is characterized by increased abundance of transcriptional repressors, particularly polycomb group (PcG) proteins. Knocking down PcG proteins precluded the induction of ischemic tolerance, whereas in an in vitro model, overexpressing the PcG proteins SCMH1 or BMI1 induced tolerance to ischemia without preconditioning. We found that PcG proteins are associated with the promoter regions of genes encoding two potassium channel proteins that show decreased abundance in ischemic-tolerant brains. Furthermore, PcG proteins decreased potassium currents in cultured neuronal cells, and knocking down potassium channels elicited tolerance without preconditioning. These findings reveal a previously unknown mechanism of neuroprotection that involves gene repressors of the PcG family.

Initial Development and Validation of a Novel Extraction Method for Quantitative Mining of the Formalin-Fixed, Paraffin-Embedded Tissue Proteome for Biomarker Investigations

Annotated formalin-fixed, paraffin-embedded (FFPE) tissue archives constitute a valuable resource for retrospective biomarker discovery. However, proteomic exploration of archival tissue is impeded by extensive formalin-induced covalent cross-linking. Robust methodology enabling proteomic profiling of archival resources is urgently needed. Recent work is beginning to support the feasibility of biomarker discovery in archival tissues, but further developments in extraction methods which are compatible with quantitative approaches are urgently needed. We report a cost-effective extraction methodology permitting quantitative proteomic analyses of small amounts of FFPE tissue for biomarker investigation. This surfactant/heat-based approach results in effective and reproducible protein extraction in FFPE tissue blocks. In combination with a liquid chromatography−mass spectrometry-based label-free quantitative proteomics methodology, the protocol enables the robust representative and quantitative analyses of the archival proteome. Preliminary validation studies in renal cancer tissues have identified typically 250−300 proteins per 500 ng of tissue with 1D LC−MS/MS with comparable extraction in FFPE and fresh frozen tissue blocks and preservation of tumor/normal differential expression patterns (205 proteins, r = 0.682; p < 10−15). The initial methodology presented here provides a quantitative approach for assessing the potential suitability of the vast FFPE tissue archives as an alternate resource for biomarker discovery and will allow exploration of methods to increase depth of coverage and investigate the impact of preanalytical factors.

Ion mobility separation coupled with MS detects two structural states of Alzheimer's disease Aβ1-40 peptide oligomers.

Mounting evidence points to the soluble oligomers of amyloid β (Aβ) peptide as important neurotoxic species in Alzheimers disease, causing synaptic dysfunction and neuronal injury, and finally leading to neuronal death. The mechanism of the Aβ peptide self-assembly is still under debate. Here, Aβ1-40 peptide oligomers were studied using mass spectrometry combined with ion mobility spectrometry, which allowed separation of the signals of numerous oligomers and measurement of their collisional cross-section values (Ω). For several oligomers, at least two different species of different Ω values were detected, indicating the presence of at least two families of conformers: compact and extended. The obtained results are rationalized by a set of molecular models of Aβ1-40 oligomer structure that provided a very good correlation between the experimental and theoretical Ω values, both for the compact and the extended forms. Our results indicate that mass spectrometry detects oligomeric species that are on-pathway in the process of fibril formation or decay, but also alternative structures which may represent off-pathway evolution of oligomers.

Enhanced lipid isomer separation in human plasma using reversed-phase UPLC with ion-mobility/high-resolution MS detection

An ultraperformance LC (UPLC) method for the separation of different lipid molecular species and lipid isomers using a stationary phase incorporating charged surface hybrid (CSH) technology is described. The resulting enhanced separation possibilities of the method are demonstrated using standards and human plasma extracts. Lipids were extracted from human plasma samples with the Bligh and Dyer method. Separation of lipids was achieved on a 100 × 2.1 mm inner diameter CSH C18 column using gradient elution with aqueous-acetonitrile-isopropanol mobile phases containing 10 mM ammonium formate/0.1% formic acid buffers at a flow rate of 0.4 ml/min. A UPLC run time of 20 min was routinely used, and a shorter method with a 10 min run time is also described. The method shows extremely stable retention times when human plasma extracts and a variety of biofluids or tissues are analyzed [intra-assay relative standard deviation (RSD) <0.385% and <0.451% for 20 and 10 min gradients, respectively (n = 5); interassay RSD <0.673% and <0.763% for 20 and 10 min gradients, respectively (n = 30)]. The UPLC system was coupled to a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer, equipped with a traveling wave ion-mobility cell. Besides demonstrating the separation for different lipids using the chromatographic method, we demonstrate the use of the ion-mobility MS platform for the structural elucidation of lipids. The method can now be used to elucidate structures of a wide variety of lipids in biological samples of different matrices.

Ion Mobility Mass Spectrometry Analysis of Human Glycourinome

Complex carbohydrates are macromolecules biosynthesized in nontemplate-type processes, bearing specific glycoepitopes involved in crucial recognition processes such as cell differentiation and cell-cell interactions. Chemical structure of single components in complex mixtures can be analyzed by mass spectrometry for determination of the size and sequence of monosaccharides involved, branching patterns, and substitution by fucose and sialic acids. For de novo identification of glycoforms in human urinome containing N- and O-free and amino acid-linked oligosaccharides, a novel method of ion mobility tandem mass spectrometry followed by computer-assisted assignment is described. Distinct patterns of ions nested specifically by their m/z values and their drift time are observed by IMS-MS. An additional peak capacity for identification of time-separated m/z values in the IMS TOF MS mode for differentiation of singly, doubly, and triply charged molecular ion species by ion mobility separation contributes to significant reduction of carbohydrate complexity in a given mass window. Profiling of glycoforms from human urinome represents a highly efficient approach for biomarker discovery and differential glycotarget identification, demonstrating potential for diagnosis of human diseases, as for congenital disorders of glycosylation.