Laser Cleavable Probes-Based Cell Surface Engineering for in Situ Sialoglycoconjugates Profiling by Laser Desorption/Ionization Mass Spectrometry

Abstract

Cell-surface sialoglycoconjugates (sialoglycoproteins and sialoglycolipids) play important roles in cell-cell interactions and related tumor metastasis process. Although there have been some analytical methods to evaluate the sialoglycoconjugates, an effective method providing both qualitative and quantitative information is still deficient. Here we establish an extraction-free, sensitive, and high-throughput platform to realize in situ detection of the cell-surface sialoglycoconjugates on various cell lines, e.g., cancer and normal cells by laser desorption/ionization mass spectrometry (LDI MS). In this proposal, azide groups were introduced into the ends of cell-surface sialoglycoconjugates by the biorthogonal method, and then the sialoglycoconjugates were armed with a laser-cleavable probe (Tphsene) through click chemistry. We can easily get the probes signal under laser irradiation, which reflected the presence of cell-surface sialoglycoconjugates. Different cell lines were discriminated simultaneously, and the LDI relative quantification agreed with fluorescent results. Besides, a linear quantitation relationship in the range of 100 fmol to 100 pmol was obtained with a designed and synthesized internal standard (phTsane) added. A detection limit of 5 fmol was obtained with good reproducibility. Based on the quantitative and high-throughput ability, we conducted pharmacodynamics study of drug (tunicamycin) on cancer cells. In addition, we found the tag was safe from sweet-spot effect of matrix adding. The simultaneous detection of sialoglycoconjugates and metabolites was therefore achieved. We believe that this laser cleavable probes-based cell-surface engineering for sialoglycoconjugates platform means great significance to diagnosis, prognosis, and therapeutic purposes. Besides, this strategy can be applied to other glycoconjugates which is hard to detect and the related disease processes when more corresponding chemically modified sugar substrates and exact biorthogonal reactions are developed.

Selection of Internal Standards for Quantitative Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis Based on Correlation Coefficients

“…For the first one, several groups developed different ways to enhance the homogeneity of the target surface. 5−18 For example, Thomas et al reported that desorption/ionization on silicon (DIOS) allows for the analysis of related small molecules in the absence of matrix with a good reproducibility. 9 Nie et al used carbon nanodots (CDs) with excellent water solubility as the matrix for quantified MALDI-TOF MS analysis of glucose in serum.…”

Carbocation Footprinting of Soluble and Transmembrane Proteins

“…22 They can be also directly observed in mass spectrometers where their lifetime is increased in vacuum and the concentration of nucleophiles is low. 23 Carbocations are strong electrophiles that should react with the nucleophilic residues in proteins. There is only one reported footprinter based on a carbocation, that is, the Koshland's reagent that labels W via an S N 1 reaction.…”

High-Throughput Single-Cell Immunoassay in the Cellular Native Environment Using Online Desalting Dual-Spray Mass Spectrometry

“…35−37 For example, the cell surface proteins labeled by trityl sulfane probes have been successfully detected in situ by MSIAs. 36 More small molecules with MS sensitivity and extensible structures, including polyethylene glycols, 31 quaternary ammonium compounds, 34 and rhodamine derivatives, 37 are usually served as mass tags. We previously developed the rhodamine-based mass tags (RMTs) that achieved 10 7 -fold signal amplification 34 and have been applied to the in situ detection of proteins on cell surfaces at a single-cell level by mass cytometry.…”

“…However, the intact protein detection is always challenging with ESI-MS because they suffer from low ionization efficiency even without salt interference, let alone the in situ detection. An alternative to achieve high sensitivity and in situ detection of proteins is the mass spectrometry-based immunoassay (MSIA), which alters the detection of protein to that of labeled mass tags with high ionization efficiency. ,− Benefitting from signal amplification of mass tags, MSAIs enable the highly sensitive protein detection with detection limits of attomole to zeptomole. , Furthermore, the MSIAs dispense with the protein isolation or extraction, and proteins on cell surfaces can be analyzed in situ. − For example, the cell surface proteins labeled by trityl sulfane probes have been successfully detected in situ by MSIAs .…”

“…An alternative to achieve high sensitivity and in situ detection of proteins is the mass spectrometry-based immunoassay (MSIA), which alters the detection of protein to that of labeled mass tags with high ionization efficiency. ,− Benefitting from signal amplification of mass tags, MSAIs enable the highly sensitive protein detection with detection limits of attomole to zeptomole. , Furthermore, the MSIAs dispense with the protein isolation or extraction, and proteins on cell surfaces can be analyzed in situ. − For example, the cell surface proteins labeled by trityl sulfane probes have been successfully detected in situ by MSIAs . More small molecules with MS sensitivity and extensible structures, including polyethylene glycols, quaternary ammonium compounds, and rhodamine derivatives, are usually served as mass tags.…”