Integrated Top-down/Bottom-up Proteomics for the Characterization of Protein Isoforms

Presented by: Ljiljana Paša-Tolić, Ph.D., Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory

Description: Obtaining extended sequence coverage of intact proteins and characterizing multiple post-translational modification (PTM) sites can pose key technical challenges. In this webinar, we describe how we have implemented Advion’s TriVersa NanoMate® and RePlay® technologies to address these issues using an integrated top-down/bottom-up approach.

We demonstrate how the TriVersa NanoMate’s capabilities of chip-based electrospray ionization (ESI) and fraction collection are used to profile isoforms, further investigate targeted proteins at the intact level using ECD or CID, perform bottom-up proteomics on a collected fraction to get confident protein identifications, perform targeted MS/MS on particular fractions of interest, and better characterize where PTMs may have occurred.

We also discuss how RePlay simplifies the data analysis process by allowing you to see the correlation between the intact protein and the observed peptides, even when you are sample limited. Using RePlay, we incorporated an on-line digestion strategy with the integrated top-down/bottom-up approach. This technique allows you to implement LC-MS/MS during both the intact run and a secondary RePlay run with digestion, so you can see the correlation in time between the observed parent ions and the digested ions as well as the MS/MS of the fragment ions.

Structure of Triplex DNA in the Gas Phase

Annalisa Arcella, Guillem Portella, Maria Luz Ruiz, Ramon Eritja, Marta Vilaseca, Valérie Gabelica, and Modesto Orozco
Journal of the American Chemical Society 2012 134 (15), 6596-6606

Shotgun lipidomics on a LTQ Orbitrap mass spectrometer by successive switching between acquisition polarity modes

Authors: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden University of Technology, Germany; Advion Biosciences Ltd., United Kingdom

Abstract

Top–down shotgun lipidomics relies on direct infusion of total lipid extracts into a high‐resolution tandem mass spectrometer and implies that individual lipids are recognized by their accurately determined m/z. Lipid ionization efficiency and detection specificity strongly depend on the acquisition polarity, and therefore it is beneficial to analyze lipid mixtures in both positive and negative modes. Hybrid LTQ Orbitrap mass spectrometers are widely applied in top–down lipidomics; however, rapid polarity switching was previously unfeasible because of the severe and immediate degradation of mass accuracy. Here, we report on a method to rapidly acquire high‐resolution spectra in both polarity modes with sub‐ppm mass accuracy and demonstrate that it not only simplifies and accelerates shotgun lipidomics analyses but also improves the lipidome coverage because more lipid classes and more individual species within each class are recognized. In this way, shotgun analysis of total lipid extracts of human blood plasma enabled to quantify 222 species from 15 major lipid classes within a 7 min acquisition cycle.

Nano-ESI analysis and on-line polarity switching were accomplished using the Advion TriVersa NanoMate.