Brian H. Clowers

A Serum Glycomics Approach to Breast Cancer Biomarkers

Because the glycosylation of proteins is known to change in tumor cells during the development of breast cancer, a glycomics approach is used here to find relevant biomarkers of breast cancer. These glycosylation changes are known to correlate with increasing tumor burden and poor prognosis. Current antibody-based immunochemical tests for cancer biomarkers of ovarian (CA125), breast (CA27.29 or CA15-3), pancreatic, gastric, colonic, and carcinoma (CA19-9) target highly glycosylated mucin proteins. However, these tests lack the specificity and sensitivity for use in early detection. This glycomics approach to find glycan biomarkers of breast cancer involves chemically cleaving oligosaccharides (glycans) from glycosylated proteins that are shed or secreted by breast cancer tumor cell lines. The resulting free glycan species are analyzed by MALDI-FT-ICR MS. Further structural analysis of the glycans can be performed in FTMS through the use of tandem mass spectrometry with infrared multiphoton dissociation. Glycan profiles were generated for each cell line and compared. These methods were then used to analyze sera obtained from a mouse model of breast cancer and a small number of serum samples obtained from human patients diagnosed with breast cancer or patients with no known history of breast cancer. In addition to the glycosylation changes detected in mice as mouse mammary tumors developed, glycosylation profiles were found to be sufficiently different to distinguish patients with cancer from those without. Although the small number of patient samples analyzed so far is inadequate to make any legitimate claims at this time, these promising but very preliminary results suggest that glycan profiles may contain distinct glycan biomarkers that may correspond to glycan “signatures of cancer.”

Profile of Native N-linked Glycan Structures from Human Serum Using High Performance Liquid Chromatography on a Microfluidic Chip and Time-of-Flight Mass Spectrometry

Protein glycosylation involves the addition of monosaccharides in a stepwise process requiring no glycan template. Therefore, identifying the numerous glycoforms, including isomers, can help elucidate the biological function(s) of particular glycans. A method to assess the diversity of the N‐linked oligosaccharides released from human serum without derivatization has been developed using on‐line nanoLC and high resolution TOF MS. The N‐linked oligosaccharides were analyzed with MALDI FT‐ICR MS and microchip LC MS (HPLC–Chip/TOF MS). Two microfluidic chips were employed, the glycan chip (40 nL enrichment column, 43×0.075 mm2 i.d. analytical column) and the high capacity chip (160 nL enrichment column, 140×0.075 mm2 i.d. analytical column), both with graphitized carbon as the stationary phase. Both chips offered good sensitivity and reproducibility in separating a heterogeneous mixture of neutral and anionic oligosaccharides between injections. Increasing the length and volume of the enrichment and the analytical columns improved resolution of the peaks. Complex type N‐linked oligosaccharides were the most abundant oligosaccharides in human serum accounting for ∼96% of the total glycans identified, while hybrid and high mannose type oligosaccharides comprise the remaining ∼4%.

Ion mobility-mass spectrometry analysis of isomeric carbohydrate precursor ions

The rapid separation of isomeric precursor ions of oligosaccharides prior to their analysis by mass spectrometry to the nth power (MSn) was demonstrated using an ambient pressure ion mobility spectrometer (IMS) interfaced with a quadrupole ion trap. Separations were not limited to specific types of isomers; representative isomers differing solely in the stereochemistry of sugars, in their anomeric configurations, and in their overall branching patterns and linkage positions could be resolved in the millisecond time frame. Physical separation of precursor ions permitted independent mass spectra of individual oligosaccharide isomers to be acquired to at least MS3, the number of stages of dissociation limited only practically by the abundance of specific product ions. IMS–MSn analysis was particularly valuable in the evaluation of isomeric oligosaccharides that yielded identical sets of product ions in tandem mass spectrometry experiments, revealing pairs of isomers that would otherwise not be known to be present in a mixture if evaluated solely by MS dissociation methods alone. A practical example of IMS–MSn analysis of a set of isomers included within a single high-performance liquid chromatography fraction of oligosaccharides released from bovine submaxillary mucin is described.

Enhanced ion utilization efficiency using an electrodynamic ion funnel trap as an injection mechanism for ion mobility spectrometry.

Conventional ion mobility spectrometers that sample ion packets from continuous sources have traditionally been constrained by an inherently low duty cycle. As such, ion utilization efficiencies have been limited to <1% in order to maintain instrumental resolving power. Using a modified electrodynamic ion funnel, we demonstrated the ability to accumulate, store, and eject ions in conjunction with ion mobility spectrometry (IMS), which elevated the charge density of the ion packets ejected from the ion funnel trap (IFT) and provided a considerable increase in the overall ion utilization efficiency of the IMS instrument. A 7-fold increase in signal intensity was revealed by comparing continuous ion beam current with the amplitude of the pulsed ion current in IFT-IMS experiments using a Faraday plate. Additionally, we describe the IFT operating characteristics using a time-of-flight mass spectrometer attached to the IMS drift tube.

Analytical Performance of Immobilized Pronase for Glycopeptide Footprinting and Implications for Surpassing Reductionist Glycoproteomics

A fully developed understanding of protein glycosylation requires characterization of the modifying oligosaccharides, elucidation of their covalent attachment sites, and determination of the glycan heterogeneity at specific sites. Considering the complexity inherent to protein glycosylation, establishing these features for even a single protein can present an imposing challenge. To meet the demands of glycoproteomics, the capability to screen far more complex systems of glycosylated proteins must be developed. Although the proteome wide examination of carbohydrate modification has become an area of keen interest, the intricacy of protein glycosylation has frustrated the progress of large-scale, systems oriented research on site-specific protein-glycan relationships. Indeed, the analytical obstacles in this area have been more instrumental in shaping the current glycoproteomic paradigm than have the diverse functional roles and ubiquitous nature of glycans. This report describes the ongoing development and analytically salient features of bead immobilized pronase for glycosylation site footprinting. The present work bears on the ultimate goal of providing analytical tools capable of addressing the diversity of protein glycosylation in a more comprehensive and efficient manner. In particular, this approach has been assessed with respect to reproducibility, sensitivity, and tolerance to sample complexity. The efficiency of pronase immobilization, attainable pronase loading density, and the corresponding effects on glycoprotein digestion rate were also evaluated. In addition to being highly reproducible, the immobilized enzymes retained a high degree of proteolytic activity after repeat usage for up to 6 weeks. This method also afforded a low level of chemical background and provided favorable levels of sensitivity with respect to traditional glycoproteomic strategies. Thus, the application of immobilized pronase shows potential to contribute to the advancement of more comprehensive glycoproteomic research methods that are capable of providing site-specific glycosylation and microheterogeneity information across many proteins.

Factors that influence fragmentation behavior of N-linked glycopeptide ions.

The investigation of site-specific glycosylation is essential for further understanding the many biological roles that glycoproteins play; however, existing methods for characterizing site-specific glycosylation either are slow or yield incomplete information. Mass spectrometry (MS) is being applied to investigate site-specific glycosylation with bottom-up proteomic type strategies. When using these approaches, tandem mass spectrometry techniques are often essential to verify glycopeptide composition, minimize false positives, and investigate structure. The fragmentation behavior of glycopeptide ions has previously been investigated with multiple techniques including collision induced dissociation (CID), infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD); however, due to the almost exclusive analysis of multiply protonated tryptic glycopeptide ions, some dissociation behaviors of N-linked glycopeptide ions have not been fully elucidated. In this study, IRMPD of N-linked glycopeptides has been investigated with a focus on the effects of charge state, charge carrier, glycan composition, and peptide composition. Each of these parameters was shown to influence the fragmentation behavior of N-linked glycopeptide ions. For example, in contrast to previously reported accounts that IRMPD results only in glycosidic bond cleavage, the fragmentation of singly protonated glycopeptide ions containing a basic amino acid residue almost exclusively resulted in peptide backbone cleavage. The fragmentation of the doubly protonated glycopeptide ion exhibited fragmentation similar to that previously reported; however, when the same glycopeptide was sodium coordinated, a previously inaccessible series of glycan fragments were observed. Molecular modeling calculations suggest that differences in the site of protonation and metal ion coordination may direct glycopeptide ion fragmentation.

Dynamically Multiplexed Ion Mobility Time-of-Flight Mass Spectrometry

Ion mobility spectrometry-time-of-flight mass spectrometry (IMS-TOFMS) has been increasingly used in analysis of complex biological samples. A major challenge is to transform IMS-TOFMS to a high-sensitivity, high-throughput platform, for example, for proteomics applications. In this work, we have developed and integrated three advanced technologies, including efficient ion accumulation in an ion funnel trap prior to IMS separation, multiplexing (MP) of ion packet introduction into the IMS drift tube, and signal detection with an analog-to-digital converter, into the IMS-TOFMS system for the high-throughput analysis of highly complex proteolytic digests of, for example, blood plasma. To better address variable sample complexity, we have developed and rigorously evaluated a novel dynamic MP approach that ensures correlation of the analyzer performance with an ion source function and provides the improved dynamic range and sensitivity throughout the experiment. The MP IMS-TOFMS instrument has been shown to reliably detect peptides at a concentration of 1 nM in the presence of a highly complex matrix, as well as to provide a 3 orders of magnitude dynamic range and a mass measurement accuracy of better than 5 ppm. When matched against human blood plasma database, the detected IMS-TOF features were found to yield approximately 700 unique peptide identifications at a false discovery rate (FDR) of approximately 7.5%. Accounting for IMS information gave rise to a projected FDR of approximately 4%. Signal reproducibility was found to be greater than 80%, while the variations in the number of unique peptide identifications were <15%. A single sample analysis was completed in 15 min that constitutes almost 1 order of magnitude improvement compared to a more conventional LC-MS approach.

Nano-LC–MS/MS of Glycopeptides Produced by Nonspecific Proteolysis Enables Rapid and Extensive Site-Specific Glycosylation Determination

Given the biological importance of glycosylation on proteins, the identification of protein glycosylation sites is integral to understanding broader biological structure and function. Unfortunately, the determination of the microheterogeneity at the site of glycosylation still remains a significant challenge. Nanoflow liquid chromatography with tandem mass spectrometry provides both separation of glycopeptides and the ability to determine glycan composition and site-specific glycosylation. However, because of the size of glycopeptides, they are not often amenable to tandem MS. In this work, proteins are digested with multiple proteases to produce glycopeptides that are of suitable size for tandem MS analysis. The conditions for collision-induced dissociation are optimized to obtain diagnostic ions that maximize glycan and peptide information. The method is applied to glycoproteins with contrasting glycans and multiple sites of glycosylation and identifies multiple glycan compositions at each individual glycosylation site. This method provides an important improvement in the routine determination of glycan microheterogeneity by mass spectrometry.

On-line digestion system for protein characterization and proteome analysis

An efficient on-line digestion system that reduces the number of sample manipulation steps has been demonstrated for high-throughput proteomics. By incorporating a pressurized sample loop into a liquid chromatography-based separation system, both sample and enzyme (e.g., trypsin) can be simultaneously introduced to produce a complete, yet rapid digestion. Both standard proteins and a complex Shewanella oneidensis global protein extract were digested and analyzed using the automated online pressurized digestion system coupled to an ion mobility time-of-flight mass spectrometer, an ion trap mass spectrometer, or both. The system denatured, digested, and separated product peptides in a manner of minutes, making it amenable to on-line high-throughput applications. In addition to simplifying and expediting sample processing, the system was easy to implement and no cross-contamination was observed among samples. As a result, the online digestion system offers a powerful approach for high-throughput screening of proteins that could prove valuable in biochemical research (rapid screening of protein-based drugs).

Direct Real-Time Detection of RDX Vapors Under Ambient Conditions

The results in this manuscript represent a demonstration of RDX vapor detection in real time at ambient temperature without sample preconcentration. The detection of vapors from the low volatility explosive compound RDX was achieved through selective atmospheric pressure chemical ionization using nitrate reactant ions (NO(3)(-)) and NO(3)(-)·HNO(3) adducts generated in an electrical discharge source. The RDX vapors were ionized in a reaction region, which provided a variable (up to several seconds) reaction time. The reaction times were controlled either by flow in an atmospheric flow tube (AFT) or by an electric field in an atmospheric drift tube (ADT). Both AFT and ADT were interfaced to a quadrupole mass spectrometer for ion detection and identification. Recorded signals were observed for RDX concentrations below 25 ppq using selected ion monitoring (SIM) of the RDX-nitrate adduct at m/z 284.