Session MOD. There are 4 abstracts in this session.


Data-independent acquisition combined with parallel accumulation – serial fragmentation combined (diaPASEF): Bottom-up proteomics with increased ion usage

Ben Collins

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A View of Multiscale Protein Organization and Function in Cancer

Joe Gray

The talk will focus on recent work using multiplex immunofluorescence microscopy, super resolution fluorescence microscopy and 3D scanning electron microscopy to explore the multiscale proteomic organization of human cancers with emphasis on elucidation of functional inter- and intercellular interactions. The interactions will be presented in the context of treatment with drugs that modulate the interactions.
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Session: TECHNOLOGY: ADVANCES IN TECHNOLOGY, time: 3:50 pm - 4:05 PM

New Directions for IR-MALDESI Mass Spectrometry Imaging: From Cells, Bones and Neurotransmitters to Drift Tube Ion Mobility

David Muddiman; Crystal Pace; Sitora Khodjaniyazova; Caleb Bagley; Anqi Tu; Hongxia Bai; James Dodds; Erin Baker
NC State University, Raleigh, NC

The mass spectrometry imaging field is moving into the realm of clinical analysis and this is largely based on MALDI and DESI ionization methods. We have developed a novel ionization method called Infrared Matrix Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) which can be readily adaptable to many different sample types.  Here, we demonstrate the effectiveness of IR-MALDESI for a wide-variety of applications and new directions using different mass spectrometry platform technologies. Specifically, a home-built IR-MALDESI sources has been coupled to high resolution accurate mass (Orbitrap) and drift tube ion mobility time-of-flight mass spectrometers.  A diverse array of sample types will be presented from HTS/HCS, bones and diverse array of tissues.  For HTS/HCS screening applications, zero sample preparation is required from very complex mixtures with the throughput of 2 samples per second.  For bones, we developed a method that does not require decalcification prior to imaging which is critical because HCl will degrade/alter molecular information present in the sample.  Finally, a wide range of ongoing studies will be presented including anti-malaria medications, HIV drugs and their co-localization with viral RNA and immune cells, how a fetus responds to exposure to fire retardants.  IR-MALDESI has demonstrated to be extraordinarily adaptable to different sample types and requires no sample preparation.  This has allowed us to carry out mass spectrometry imaging for a wide-range of applications that relate to human health and exposure. 

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Isobaric quantitative protein interaction reporter technology for comparative interactome studies

Juan Chavez; Andrew Keller; Jared Mohr; Jimmy Eng; James Bruce
University of Washington, Seattle, WA

Chemical cross-linking with mass spectrometry (XL-MS) has emerged as a useful tool for the large scale in situ study of protein structures and interactions from complex biological samples including intact cells and tissues.  Quantifying cross-linked peptides provides unique information on protein conformational and interaction changes resulting from perturbations such as drug treatment and disease state.  Previous quantitative XL-MS studies have relied on incorporation of stable isotopes into the cross-linker (primarily deuterium), metabolic labeling of amino acid residues or incorporation of additional labeling steps.  Here we report on the development of isobaric quantitative Protein Interaction Reporter (iqPIR) technology which utilizes stable isotopes selectively incorporated into the cross-linker design, allowing for cross-linked peptide pairs originating from different samples to have exactly the same mass in MS1 measurements, yet display distinct quantitative isotope signatures in tandem MS.  This affords multiple benefits over other existing quantitative XL-MS strategies including increased signal-to-noise due to additive contributions of MS1 signals, generation of multiple fragment ions carrying quantitative information in a single tandem MS spectrum, avoidance of chromatographic alignment and peak assignment problems, elimination of retention time shifts between isotope partners as commonly observed with deuterium, applicability to systems where metabolic labeling is impractical, and potential for multiplexed quantitation. 

We will present details on the molecular design and synthesis of iqPIR cross-linkers, development of custom informatics capabilities, and their application to model systems as well as pharmacological-induced interactome changes in human cultured cells.  Data will be compared and contrasted with a previous quantitative cross-linking study using SILAC to evaluate the effects of the Hsp90 inhibitor 17-AAG on the interactome.  Data from these initial applications of iqPIR indicate a high level of quantitative information with over 90% of cross-linked peptides identified producing quantitative values with excellent accuracy and precision. 

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