Analysis of Glycopeptides by HPLC MS and Detection of Leukemic Cells by Microfluidics using Time Delayed Integration Spectral Flow Cytometry

Abstract

Protein glycosylation drives many biological processes and serves as marker for disease; therefore, the development of tools to study glycosylation is an essential and growing area of research. Mass spectrometry can be used to identify both the glycans of interest and the glycosylation sites to which those glycans are attached, when proteins are proteolytically digested and their glycopeptides are analyzed by a combination of high-resolution mass spectrometry (MS) and tandem mass spectrometry (MS/MS) methods. One major challenge in these experiments is collecting the requisite MS/MS data. The digested glycopeptides are often present in complex mixtures and in low abundance, and the most commonly used approach to collect MS/MS data on these species is data-dependent acquisition (DDA), where only the most intense precursor ions trigger MS/MS. DDA results in limited glycopeptide coverage. Semi-targeted data acquisition is an alternative experimental approach that can alleviate this difficulty. However, due to the massive heterogeneity of glycopeptides, it is not obvious how to expediently generate inclusion lists for these types of analyses. To solve this problem, we developed the software tool GlycoPep MassList, which can be used to generate inclusion lists for liquid chromatography tandem-mass spectrometry (LC-MS/MS) experiments. The utility of the software was tested by conducting comparisons between semi-targeted and untargeted data-dependent analysis experiments on a variety of proteins, including IgG, a protein whose glycosylation must be characterized during its production as a biotherapeutic. When the GlycoPep MassList software was used to generate inclusion lists for LC-MS/MS experiments, more unique glycopeptides were selected for fragmentation. Generally, ∼30 % more unique glycopeptides can be analyzed per protein, in the simplest cases, with low background. In cases where background ions from proteins or other interferents are high, usage of an inclusion list is even more advantageous. The software is free and publicly accessible. In another research project, we describe a unique flow cytometer (TDI SFC) that combines the high spectral resolution of spectral flow cytometry (SFC) with a CCD operated in time-delayed integration (TDI) mode for the automated immunophenotyping of rare, low abundant cells. A microfluidic device providing 1-D focusing was used to sheath cells through a 488 nm laser excitation beam. Using epi-illumination, a spectrograph was included into the emission optical path to spectrally disperse the emission along one axis of a CCD camera. The parallel shift rate of the CCD was synchronized to the cell travel through the field-of-view, which was defined by the excitation volume. This TDI SFC format allowed the CCD shutter to remain open during signal acquisition and as such, the duty cycle was ~100% allowing for rare cells to not be missed. Fluorescent calibration beads were used to optimize synchronization of the CCD’s TDI clocking with the sheathed cell velocity, TDI SFC sensitivity, excitation power intensity, epi-illumination objective’s numerical aperture, and total integration time. TDI integrated signals of 106 counts at a signal-to-noise ratio (SNR) of 610 for beads corresponding to a load of 4×105 antibodies per bead was achieved. Additionally, we evaluated the multiplexing capabilities by performing spectral deconvolution. Finally, a proof-of-concept application was undertaken to immunophenotype rare cells, specifically leukemic cells circulating in the blood of patients with B-cell acute lymphoblastic leukemia (B-ALL) for monitoring measurable residual disease (MRD). A B-ALL cell line was stained against a leukemic marker (TdT) to successfully discriminate TdT(+) circulating leukemic cells from normal B cells at very low cell counts (≤100 cells).