Revolutions in Lipid Isomer Resolution: Application of Ultrahigh-Resolution Ion Mobility to Reveal Lipid Diversity.

Many families of lipid isomers remain unresolved by contemporary liquid chromatography-mass spectrometry approaches, leading to a significant underestimation of the structural diversity within the lipidome. While ion mobility coupled to mass spectrometry has provided an additional dimension of lipid isomer resolution, some isomers require a resolving power beyond the capabilities of conventional platforms. Here, we present the application of high-resolution traveling-wave ion mobility for the separation of lipid isomers that differ in (i) the location of a single carbon-carbon double bond, (ii) the stereochemistry of the double bond (cis or trans), or, for glycerolipids, (iii) the relative substitution of acyl chains on the glycerol backbone (sn-position). Collisional activation following mobility separation allowed identification of the carbon-carbon double-bond position and sn-position, enabling confident interpretation of variations in mobility peak abundance. To demonstrate the applicability of this method, double-bond and sn-position isomers of an abundant phosphatidylcholine composition were resolved in extracts from a prostate cancer cell line and identified by comparison to pure isomer reference standards, revealing the presence of up to six isomers. These findings suggest that ultrahigh-resolution ion mobility has broad potential for isomer-resolved lipidomics and is attractive to consider for future integration with other modes of ion activation, thereby bringing together advanced orthogonal separations and structure elucidation to provide a more complete picture of the lipidome.

Global Profiling of Metabolite and Lipid Soluble Microbial Products in Anaerobic Wastewater Reactor Supernatant Using UPLC-MSE.

Identification of soluble microbial products (SMPs) released during bacterial metabolism in mixed cultures in bioreactors is essential to understanding fundamental mechanisms of their biological production. SMPs constitute one of the main foulants (together with colloids and bacterial flocs) in membrane bioreactors widely used to treat and ultimately recycle wastewater. More importantly, the composition and origin of potentially toxic, carcinogenic, or mutagenic SMPs in renewable/reused water supplies must be determined and controlled. Certain classes of SMPs have previously been studied by GC-MS, LC-MS, and MALDI-ToF MS; however, a more comprehensive LC-MS-based method for SMP identification is currently lacking. Here we develop a UPLC-MS approach to profile and identify metabolite SMPs in the supernatant of an anaerobic batch bioreactor. The small biomolecules were extracted into two fractions based on their polarity, and separate methods were then used for the polar and nonpolar metabolites in the aqueous and lipid fractions, respectively. SMPs that increased in the supernatant after feed addition were identified primarily as phospholipids, ceramides, with cardiolipins in the highest relative abundance, and these lipids have not been previously reported in wastewater effluent.

Observations on the detection of b- and y-type ions in the collisionally activated decomposition spectra of protonated peptides.

Tandem mass spectrometric data from peptides are routinely used in an unsupervised manner to infer product ion sequence and hence the identity of their parent protein. However, significant variability in relative signal intensity of product ions within peptide tandem mass spectra is commonly observed. Furthermore, instrument-specific patterns of fragmentation are observed, even where a common mechanism of ion heating is responsible for generation of the product ions. This information is currently not fully exploited within database searching strategies; this motivated the present study to examine a large dataset of tandem mass spectra derived from multiple instrumental platforms. Here, we report marked global differences in the product ion spectra of protonated tryptic peptides generated from two of the most common proteomic platforms, namely tandem quadrupole-time-of-flight and quadrupole ion trap instruments. Specifically, quadrupole-time-of-flight tandem mass spectra show a significant under-representation of N-terminal b-type fragments in comparison to quadrupole ion trap product ion spectra. Energy-resolved mass spectrometry experiments conducted upon test tryptic peptides clarify this disparity; b-type ions are significantly less stable than their y-type N-terminal counterparts, which contain strongly basic residues. Secondary fragmentation processes which occur within the tandem quadrupole-time-of-flight device account for the observed differences, whereas this secondary product ion generation does not occur to a significant extent from resonant excitation performed within the quadrupole ion trap. We suggest that incorporation of this stability information in database searching strategies has the potential to significantly improve the veracity of peptide ion identifications as made by conventional database searching strategies.

Observation of heterogeneous gene products by FT-ICR MS.

The ability of FT-ICR MS to resolve isotopic variants of intact proteins for each of the charge states formed by electrospray ionization offers a sensitive, rapid method for detecting "low mass" heterogeneity, where this is defined as the presence of structural variants differing in mass by 2 Da or less. Such heterogeneity may reflect biological or chemical modifications of structure or may result from the coexpression of related proteins from a multi-gene family. In the analytical approach described here, comparisons are made between observed isotopic distributions and those expected for predicted protein sequences. Close agreement is demonstrated for a homogeneous model protein, and the utility of the method has been evaluated in the study of mouse major urinary proteins (MUPs), a group of closely related sequences. Divergence of the experimental isotopic distribution from distributions predicted for known MUP sequences can be explained, in quantitative terms, by the coexpression of closely related sequences. This approach provides a facile method for the assessment of protein homogeneity and for the detection of structural variants, without recourse to proteolytic digestion and analysis of the resulting products.

Protein quantification by selective isolation and fragmentation of isotopic pairs using FT-ICR MS.

Isolation of tryptic peptide ions, along with their differentially labeled analogs derived from an artificial QconCAT protein, is performed using multiple correlated harmonic excitation fields in an FT-ICR cell. Simultaneous fragmentation of the isolated unlabeled and labeled peptide pairs using IRMPD yields specific y-series fragment ions useful for quantification. The mass increment attributed to stable isotope labeling at the C-terminus is maintained in the C-terminal fragment ions, providing multiple measurements of labeled/unlabeled intensity ratios during highly selective detection. The utility of this approach has been demonstrated in the absolute quantification of components of an unfractionated chicken muscle protein mixture.

QCAL--a novel standard for assessing instrument conditions for proteome analysis.

If proteome datasets are to be collated, shared, and merged for higher level proteome analyses, there is a need for generally accepted strategies and reagents for optimization and standardization of instrument performance. At present, there is no single protein or peptide standard set that is capable of assessing instrument performance for peptide separation and analysis in this manner. To create such a standard, we have used the recently described QconCAT methodology to generate an artificial protein, QCAL. This protein, a concatenation of tryptic peptides that is expressed in E. coli, provides a stoichiometrically controlled mixture of peptides that are amenable to analysis by all commonly used instrumentation platforms for proteomics.