Quantifying Kinase-Specific Phosphorylation Stoichiometry Using Stable Isotope Labeling In a Reverse In-Gel Kinase Assay

Despite recent advancements in large-scale phosphoproteomics, methods to quantify kinase-specific phosphorylation stoichiometry of protein substrates are lacking. We developed a method to quantify kinase-specific phosphorylation stoichiometry by combining the reverse in-gel kinase assay (RIKA) with high-resolution liquid chromatography–mass spectrometry (LC–MS). Beginning with predetermined ratios of phosphorylated to nonphosphorylated protein kinase CK2 (CK2) substrate molecules, we employed ¹⁸O-labeled adenosine triphosphate (¹⁸O-ATP) as the phosphate donor in a RIKA, then quantified the ratio of ¹⁸O- versus ¹⁶O-labeled tryptic phosphopeptide using high mass accuracy mass spectrometry (MS). We demonstrate that the phosphorylation stoichiometry determined by this method across a broad percent phosphorylation range correlated extremely well with the predicted value (correlation coefficient = 0.99). This approach provides a quantitative alternative to antibody-based methods of determining the extent of phosphorylation of a substrate pool

Ion Elevators and Escalators in Multilevel Structures for Lossless Ion Manipulations

We describe two approaches based upon ion “elevator” and “escalator” components that allow moving ions to different levels in structures for lossless ion manipulations (SLIM). Guided by ion motion simulations, we designed elevator and escalator components based upon ion current measurements providing essentially lossless transmission in multilevel designs. The ion elevator design allowed ions to efficiently bridge a 4 mm gap between levels. The component was integrated in a SLIM and coupled to a QTOF mass spectrometer using an ion funnel interface to evaluate the <i>m</i>/<i>z</i> range transmitted as compared to transmission within a level (e.g., in a linear section). The analysis of singly charged ions of <i>m</i>/<i>z</i> 600–2700 produced similar mass spectra for both elevator and straight (linear motion) components. In the ion escalator design, traveling waves (TW) were utilized to transport ions efficiently between two SLIM levels. Ion current measurements and ion mobility (IM) spectrometry analysis illustrated that ions can be transported between TW-SLIM levels with no significant loss of either ions or IM resolution. These developments provide a path for the development of multilevel designs providing, e.g., much longer IM path lengths, more compact designs, and the implementation of much more complex SLIM devices in which, e.g., different levels may operate at different temperatures or with different gases

Polysialylated N‑Glycans Identified in Human Serum Through Combined Developments in Sample Preparation, Separations, and Electrospray Ionization-Mass Spectrometry

The N-glycan diversity of human serum glycoproteins, i.e., the human blood serum N-glycome, is both complex and constrained by the range of glycan structures potentially synthesizable by human glycosylation enzymes. The known glycome, however, has been further limited by methods of sample preparation, available analytical platforms, e.g., based upon electrospray ionization-mass spectrometry (ESI-MS), and software tools for data analysis. In this report several improvements have been implemented in sample preparation and analysis to extend ESI-MS glycan characterization and to include polysialylated N-glycans. Sample preparation improvements included acidified, microwave-accelerated, PNGase F N-glycan release to promote lactonization, and sodium borohydride reduction, that were both optimized to improve quantitative yields and conserve the number of glycoforms detected. Two-stage desalting (during solid phase extraction and on the analytical column) increased sensitivity by reducing analyte signal division between multiple reducing-end-forms or cation adducts. Online separations were improved by using extended length graphitized carbon columns and adding TFA as an acid modifier to a formic acid/reversed phase gradient, providing additional resolving power and significantly improved desorption of both large and heavily sialylated glycans. To improve MS sensitivity and provide gentler ionization conditions at the source-MS interface, subambient pressure ionization with nanoelectrospray (SPIN) was utilized. When these improved methods are combined together with the Glycomics Quintavariate Informed Quantification (GlyQ-IQ) recently described (Kronewitter et al. Anal. Chem. 2014, 86, 6268−6276), we are able to significantly extend glycan detection sensitivity and provide expanded glycan coverage. We demonstrated the application of these advances in the context of the human serum glycome, and for which our initial observations included the detection of a new class of heavily sialylated N-glycans, including polysialylated N-glycans

Polysialylated N‑Glycans Identified in Human Serum Through Combined Developments in Sample Preparation, Separations, and Electrospray Ionization-Mass Spectrometry

The N-glycan diversity of human serum glycoproteins, i.e., the human blood serum N-glycome, is both complex and constrained by the range of glycan structures potentially synthesizable by human glycosylation enzymes. The known glycome, however, has been further limited by methods of sample preparation, available analytical platforms, e.g., based upon electrospray ionization-mass spectrometry (ESI-MS), and software tools for data analysis. In this report several improvements have been implemented in sample preparation and analysis to extend ESI-MS glycan characterization and to include polysialylated N-glycans. Sample preparation improvements included acidified, microwave-accelerated, PNGase F N-glycan release to promote lactonization, and sodium borohydride reduction, that were both optimized to improve quantitative yields and conserve the number of glycoforms detected. Two-stage desalting (during solid phase extraction and on the analytical column) increased sensitivity by reducing analyte signal division between multiple reducing-end-forms or cation adducts. Online separations were improved by using extended length graphitized carbon columns and adding TFA as an acid modifier to a formic acid/reversed phase gradient, providing additional resolving power and significantly improved desorption of both large and heavily sialylated glycans. To improve MS sensitivity and provide gentler ionization conditions at the source-MS interface, subambient pressure ionization with nanoelectrospray (SPIN) was utilized. When these improved methods are combined together with the Glycomics Quintavariate Informed Quantification (GlyQ-IQ) recently described (Kronewitter et al. Anal. Chem. 2014, 86, 6268−6276), we are able to significantly extend glycan detection sensitivity and provide expanded glycan coverage. We demonstrated the application of these advances in the context of the human serum glycome, and for which our initial observations included the detection of a new class of heavily sialylated N-glycans, including polysialylated N-glycans