Characterization of Phosphorylated Peptides by Electron-Activated and Ultraviolet Dissociation Mass Spectrometry: A Comparative Study with Collision-Induced Dissociation.

Mass-spectrometry-based methods have made significant progress in the characterization of post-translational modifications (PTMs) in peptides and proteins; however, room remains to improve fragmentation methods. Ideal MS/MS methods are expected to simultaneously provide extensive sequence information and localization of PTM sites and retain labile PTM groups. This collection of criteria is difficult to meet, and the various activation methods available today offer different capabilities. In order to examine the specific case of phosphorylation on peptides, we investigate electron transfer dissociation (ETD), electron-activated dissociation (EAD), and 193 nm ultraviolet photodissociation (UVPD) and compare all three methods with classical collision-induced dissociation (CID). EAD and UVPD show extensive backbone fragmentation, comparable in scope to that of CID. These methods provide diverse backbone fragmentation, producing a/x, b/y, and c/z ions with substantial sequence coverages. EAD displays a high retention efficiency of the phosphate modification, attributed to its electron-mediated fragmentation mechanisms, as observed in ETD. UVPD offers reasonable retention efficiency, also allowing localization of the PTM site. EAD experiments were also performed in an LC-MS/MS workflow by analyzing phosphopeptides spiked in human plasma, and spectra allow accurate identification of the modified sites and discrimination of isomers. Based on the overall performance, EAD and 193 nm UVPD offer alternative options to CID and ETD for phosphoproteomics.

Mass Spectrometry Strategies for O-Glycoproteomics.

Glycoproteomics has accelerated in recent decades owing to numerous innovations in the analytical workflow. In particular, new mass spectrometry strategies have contributed to inroads in O-glycoproteomics, a field that lags behind N-glycoproteomics due to several unique challenges associated with the complexity of O-glycosylation. This review will focus on progress in sample preparation, enrichment strategies, and MS/MS techniques for the identification and characterization of O-glycoproteins.

Ultraviolet Photodissociation Permits Comprehensive Characterization of O-Glycopeptides Cleaved with O-Glycoprotease IMPa.

Complete O-glycosite characterization, including identification of the peptides, localization of the glycosites, and mapping of the glycans, has been a persistent challenge in O-glycoproteomics owing to the technical challenges surrounding O-glycan analysis. Multi-glycosylated peptides pose an even greater challenge owing to their potential heterogeneity. Ultraviolet photodissociation (UVPD) can localize multiple post-translational modifications and is well-suited for the characterization of glycans. Three glycoproteins were assessed based on a strategy combining the use of O-glycoprotease IMPa and HCD-triggered UVPD for the complete characterization of O-glycopeptides. This approach localized multiple adjacent or proximal O-glycosites on individual glycopeptides and identified a previously unknown glycosite on etanercept at S218. Nine different glycoforms were characterized as a multi-glycosylated peptide from etanercept. The performance of UVPD was compared to that of HCD and EThcD for the localization of O-glycosites and the characterization of the constituent peptides and glycans.

The effect of protein expression on cancer cell capture using the Human Transferrin Receptor (CD71) as an affinity ligand.

Cancer cell detection in liquid biopsies has been a widely studied application in many microfluidic devices. The use of a common antibody, such as the epithelial cell adhesion molecule (Anti-EpCAM) or other specific antibodies, has facilitated the detection and study of many cancers. However, the use of such antibodies requires a priori knowledge of the cancer source, and many cancer subtypes are missed in screening applications. There remains a need to study a wider range of cancers that maintain the streamlined antibody approach in cell affinity separations. The Human transferrin receptor (CD71) has recently been demonstrated as a cancer cell affinity target in blood samples. CD71 expression in blood cells is low, whereas proliferating cancer cells have a higher expression of the surface protein. CD71 expression is variable with cell cycle, which can impact cell separations. In this work, we investigated the effects of cell cycle and CD71 expression on cell capture metrics. Six cancer cell lines were isolated from blood via CD71 affinity capture, with a capture efficiency and purity that varied with CD71 expression. Despite variation in CD71 expression, the affinity was sufficient to isolate cancer cells spiked into blood; under optimal conditions, CD71-based capture resulted in capture purity >80%. We conclude that CD71 affinity separations show potential as a biomarker for cancer studies without sacrificing sensitivity and selectivity, and that cancer cells can be isolated from liquid biopsies over a range of expression of the target protein.

Conversion of aldimines to secondary amines using iron-catalysed hydrosilylation.

Iron-catalyzed hydrosilylation of imines to amines using a well-defined iron complex is reported. This method employs relatively mild conditions, by reaction of imine, (EtO)3SiH in a 1 : 2 ratio in the presence of 1 mol% precatalyst ([BIAN]Fe(η6-toluene), 3, BIAN = bis(2,6-diisopropylaniline)acenaphthene) at 70 °C. A broad scope of imines was readily converted into the corresponding secondary amines without the need for precatalyst activators.