Matrix-assisted ionization vacuum (MAIV) is a novel ionization technique that generates multiply charged ions in vacuum without the use of laser ablation or high voltage. MAIV can be achieved in intermediate-vacuum and high-vacuum matrix-assisted laser desorption/ionization (MALDI) sources and electrospray ionization (ESI) sources without instrument modification. Herein, we adapt MAIV onto the MALDI-LTQ-Orbitrap XL platform for biomolecule analysis. As an attractive alternative to MALDI for in solution and in situ analysis of biomolecules, MAIV coupling to high resolution and accurate mass (HRAM) MS instrument has successfully expanded the mass detection range and improved the fragmentation efficiency due to the generation of multiply charged ions. Additionally, the softness of MAIV enables potential application in labile post-translational modification (PTM) analysis. In this study, proteins as large as 18.7 kDa were detected with up to 18 charges; intact peptides with labile PTM were well preserved during the ionization process and characterized MS/MS; peptides and proteins in complex tissue samples were detected and identified both in liquid extracts and in situ. Moreover, we demonstrated that this method facilitates MS/MS analysis with improved fragmentation efficiency compared to MALDI-MS/MS.
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) is a powerful tool to map the spatial distribution of biomolecules on tissue sections. Recent developments of hybrid MS instruments allow combination of different types of data acquisition by various mass analyzers into a single MSI analysis, which reduces experimental time and sample consumptions. Here, using the well-characterized crustacean nervous system as a test-bed, we explore the utility of high resolution and accurate mass (HRAM) MALDI Orbitrap platform for enhanced in situ characterization of the neuropeptidome with improved chemical information. Specifically, we report on a multiplex-MSI method, which combines HRAM MSI with data dependent acquisition (DDA) tandem MS analysis in a single experiment. This method enables simultaneous mapping of neuropeptide distribution, sequence validation and novel neuropeptide discovery in crustacean neuronal tissues. To enhance the dynamic range and efficiency of in situ DDA, we introduced a novel approach of fractionating full m/z range into several sub-mass ranges and embedding the setup using the multiplex-DDA-MSI scan events to generate pseudo fractionation before MS/MS scans. The division of entire m/z into multiple segments of m/z subranges for MS interrogation greatly decreased the complexity of molecular species from tissue samples and the heterogeneity of the distribution and variation of intensities of m/z peaks. By carefully optimizing the experimental conditions such as the dynamic exclusion, the multiplex-DDA-MSI approach demonstrates better performance with broader precursor coverage, less biased MS/MS scans towards high abundance molecules and improved quality of tandem mass spectra for low intensity molecular species.
The Chinese hamster ovary (CHO) cell line is a major expression system for the production of therapeutic proteins, the majority of which are glycoproteins, such as antibodies and erythropoietin (EPO). The characterization glycosylation profile of therapeutic proteins produced from engineered CHO cells and therapeutic functions, as well as side effects, are critical to understand the important roles of glycosylation. In this study, a large scale glycoproteomic workflow was established and applied to CHO-K1 cells expressing EPO. The workflow includes enrichment of intact glycopeptides from CHO-K1 cell lysate and medium using hydrophilic enrichment, fractionation of the obtained intact glycopeptides (IGPs) by basic reversed phase liquid chromatography (bRPLC), analyzing the glycopeptides using LC-MS/MS, and annotating the results by GPQuest 2.0. A total of 10 338 N-linked glycosite-containing IGPs were identified, representing 1162 unique glycosites in 530 glycoproteins, including 71 unique atypical N-linked IGPs on 18 atypical N-glycosylation sequons with an overrepresentation of the N-X-C motifs. Moreover, we compared the glycoproteins from CHO cell lysate with those from medium using the in-depth N-linked glycoproteome data. The obtained large scale glycoproteomic data from intact N-linked glycopeptides in this study is complementary to the genomic, proteomic, and N-linked glycomic data previously reported for CHO cells. Our method has the potential to monitor the production of recombinant therapeutic glycoproteins.
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has emerged as a label-free analytical tool for fast biomolecule profiling on tissue sections. Among various functional molecules, mapping neurotransmitters and related metabolites is of tremendous significance, as these compounds are critical to signaling in the central nervous system. Here, we demonstrated the use of both derivatization and reaction-free approaches that greatly reduced signal complexity and thus enabled complementary signaling molecule visualization on crab brain sections via MALDI-LTQ-Orbitrap XL platform. Pyrylium salt served as a primary amine derivatization reagent and produced prominent signal enhancement of multiple neurotransmitters, including dopamine, serotonin, γ-aminobutyric acid, and histamine that were not detected in underivatized tissues. Molecules with other functional groups, such as acetylcholine and phosphocholine, were directly imaged after matrix application. The identities of discovered neurotransmitters were verified by standards using LC-MS/MS. This study broadens our understanding of metabolic signaling in the crustacean nervous system and highlights potential of multifaceted MS techniques for unambiguous neurotransmitter characterization.
Introduced in 2000, atmospheric pressure (AP)/matrix-assisted laser desorption/ionization (MALDI) has attracted substantial attention in the mass spectrometry community due to its ease of sample introduction and handling, interchangeability with ESI source and capability of analyzing volatile species. In this study, an AP/MALDI source with ultra-high spatial resolution was coupled to a Q Exactive HF orbitrap mass spectrometer for high resolution in situ analysis by MALDI, laserspray ionization (LSI) and matrix assisted ionization (MAI) without instrument modification. LSI and MAI generated multiply charged ions, which expanded the mass detection range and improved fragmentation efficiency. Full MS, targeted MS/MS, data dependent acquisition (DDA) and parallel reaction monitoring (PRM) acquisitions were performed on peptide and protein standards, tissue extracts and tissue sections for in depth characterization of various biomolecules. High resolution full MS and MS/MS images were obtained from crustacean and rat tissues with pixel size less than 30 μm. Overall, AP/MALDI-Q-Orbitrap is a fast scanning instrument that is capable of performing multiple types of ionization and multiple acquisition modes without instrument modification. This instrument platform provides an attractive alternative to other high resolution MALDI instruments.
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