Mass Spectrometry‐Based Bottom‐Up Proteomics: Sample Preparation, LC‐MS/MS Analysis, and Database Query Strategies

2016 ◽  
Vol 86 (1) ◽  
Author(s):  
Matthew J. Wither ◽  
Kirk C. Hansen ◽  
Julie A. Reisz
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Database Query ◽  
Bottom Up ◽  
Ms Analysis

scholarly journals Germanium determination in environmental object by the method of mass spectrometry with inductively coupled plasma

2019 ◽  
Vol 85 (4) ◽  
pp. 110-113
Author(s):  
Olexandr Ponomarenko ◽  
Anatolyi Samchuk ◽  
Kateryna Vovk ◽  
Igor Shvaika ◽  
Ganna Grodzinskaya
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Inductively Coupled Plasma ◽  
Microwave Field ◽  
Antitumor Effects ◽  
Inductively Coupled ◽  
Ms Analysis ◽  
Icp Ms ◽  
Relative Standard

The analytical technologies of sample preparation of rocks and mushrooms using the microwave field for the determination of germanium by the method of mass spectrometry with inductively coupled plasma (ICP-MS analysis) have been developed. Germanium is a rare element. Germanium is homology of silicon and carbon. To date, the definition of low content of germanium in geological objects is a rather complex analytical task, which requires its concentration - extraction, co-precipitation, ion exchange. At present, the harmonious combination of the method of natural objects decomposition in the microwave field and germanium determination using ICP-MS analysis is particularly promising. Sample preparation of silicate rocks for ICP-MS determination of germanium was carried out by decomposition in a mixture of hydrofluoric, phosphate and nitric acids (5: 5: 2) in a microwave oven program at 240°C for 30 min. Sample preparation of mushrooms for ICP-MS germanium determination was carried out according to the following scheme. Initially, the dried sample was sealed in the presence of CaO, after dissolving it in a mixture of HNO3+HF+H3PO4 (6:6:1). Ge solution was extracted by Nazarenko V.A. extraction method. The developed analytical schemes have made it possible to significantly reduce the duration and labor intensity of sample preparation. The obtained solutions were analyzed using an inductively coupled plasma mass spectrometer. The developed method for determining germanium by ICP-MS analysis has been successfully tested on standard rock samples. The obtained results are in accordance with the accepted attribute, the relative standard deviation Sr ranges from 0.7-0.9. The data on the content and distribution of germanium in the Boletales fungi are obtained. They indicate wild mushrooms contain high levels of germanium, especially Boletus and Mushroom biospores. These studies are necessary because the essential properties of germanium and its compounds attract special attention of scientists today. Complementary Ge compounds which have hypotensive, bactericidal, antiviral and antitumor effects have already been synthesized.

scholarly journals Methodology to Quantify Collagen Subtypes and Crosslinks: Application in Minipig Cartilages

Cartilage ◽  
2021 ◽  
pp. 194760352110605
Author(s):  
Benjamin J. Bielajew ◽  
Jerry C. Hu ◽  
Kyriacos A. Athanasiou
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tissue Characterization ◽  
Collagen Type ◽  
Facet Joint ◽  
Type I ◽  
Collagen Types ◽  
Collagen Crosslinks ◽  
Bottom Up ◽  
Ms Analysis

Introduction This study develops assays to quantify collagen subtypes and crosslinks with liquid chromatography-mass spectrometry (LC-MS) and characterizes the cartilages in the Yucatan minipig. Methods For collagen subtyping, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed on tissues digested in trypsin. For collagen crosslinks, LC-MS analysis was performed on hydrolysates. Samples were also examined histologically and with bottom-up proteomics. Ten cartilages (femoral condyle, femoral head, facet joint, floating rib, true rib, auricular cartilage, annulus fibrosus, 2 meniscus locations, and temporomandibular joint disc) were analyzed. Results The collagen subtyping assay quantified collagen types I and II. The collagen crosslinks assay quantified mature and immature crosslinks. Collagen subtyping revealed that collagen type I predominates in fibrocartilages and collagen type II in hyaline cartilages, as expected. Elastic cartilage and fibrocartilages had more mature collagen crosslink profiles than hyaline cartilages. Bottom-up proteomics revealed a spectrum of ratios between collagen types I and II, and quantified 42 proteins, including 24 collagen alpha-chains and 12 minor collagen types. Discussion The novel assays developed in this work are sensitive, inexpensive, and use a low operator time relative to other collagen analysis methods. Unlike the current collagen assays, these assays quantify collagen subtypes and crosslinks without an antibody-based approach or lengthy chromatography. They apply to any collagenous tissue, with broad applications in tissue characterization and tissue engineering. For example, a novel finding of this work was the presence of a large quantity of collagen type III in the white-white knee meniscus and a spectrum of hyaline and fibrous cartilages.

Miniaturized sample preparation on a digital microfluidics device for sensitive bottom-up microproteomics of mammalian cells using magnetic beads and mass spectrometry-compatible surfactants

Lab on a Chip ◽  
2019 ◽  
Vol 19 (20) ◽  
pp. 3490-3498 ◽  
Author(s):  
Jan Leipert ◽  
Andreas Tholey
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Mammalian Cells ◽  
Magnetic Beads ◽  
Digital Microfluidics ◽  
Bottom Up ◽  
Polymer Surfactants

The combination of digital microfluidics and magnetic beads for removal of polymer surfactants enables sensitive LC-MS-based microproteomics analyses down to 100 mammalian cells.

scholarly journals Protocol for Decoding the Protein Composition of Whole Nucleosomes with Nuc-MS: Sample Preparation, Data Acquisition and Analysis

2021 ◽  
Author(s):  
Luis F. Schachner ◽  
Alexander S. Lee ◽  
Neil L. Kelleher
Keyword(s):  
Mass Spectrometry ◽  
Data Analysis ◽  
Mass Spectrum ◽  
Sample Preparation ◽  
Data Acquisition ◽  
Histone Modifications ◽  
Protein Composition ◽  
Ms Analysis ◽  
Level Information ◽  
Single Mass

Abstract The disassembly and digestion of nucleosome particles in current proteomics approaches forfeits correlations among histones and blurs nucleosome-level information. We developed Nuc-MS which analyzes whole nucleosomes and displays their histone modifications and variants in a single mass spectrum. In this protocol, we provide step-by-step instructions for preparation of mononucleosomes for mass spectrometry (MS) analysis, and parameter sets for Nuc-MS data acquisition and data analysis.

scholarly journals Histone sample preparation for bottom-up mass spectrometry: a roadmap to informed decisions

2021 ◽  
Author(s):  
Simon Daled ◽  
Sander Willems ◽  
Bart Van Puyvelde ◽  
Laura Corveleyn ◽  
Sigrid Verhelst ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Research Question ◽  
Stable Gene ◽  
Assessment Procedure ◽  
Bottom Up ◽  
Genome Complexity ◽  
Arginine Residues ◽  
Multicellular Organisms ◽  
Selection Of

Histone-based chromatin organization enabled eukaryotic genome complexity. This epigenetic control mechanism allowed for the differentiation of stable gene-expression and thus the very existence of multicellular organisms. This existential role in biology makes histones one of the most complexly modified molecules in the biotic world, which makes these key regulators notoriously hard to analyze. We here provide a roadmap to enable fast and informed selection of a bottom-up mass spectrometry sample preparation protocol that matches a specific research question. We therefore propose a two-step assessment procedure: (i) visualization of the coverage that is attained for a given workflow and (ii) direct alignment-between-runs to assess potential pitfalls at the ion level. To illustrate the applicability, we compare four different sample preparation protocols, while adding a new enzyme to the toolbox, i.e., RgpB (GingisREX, Genovis), an endoproteinase that selectively and efficiently cleaves at the c-terminal end of arginine residues.

scholarly journals An Effective and Efficient Sample Preparation Method for 2-Methyl-Isoborneol and Geosmin in Fish and Their Analysis by Gas Chromatography-Mass Spectrometry

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Liang-liang Tian ◽  
Feng Han ◽  
Essy Kouadio Fodjo ◽  
Wenlei Zhai ◽  
Xuan-Yun Huang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Sample Preparation ◽  
Large Scale ◽  
Solid Phase ◽  
Gas Chromatography Mass Spectrometry ◽  
Chromatography Mass Spectrometry ◽  
Ms Analysis ◽  
Fish Samples

The intensive aquaculture strategy and recirculating aquaculture system often lead to the production of off-flavor compounds such as 2-methyl-isoborneol (2-MIB) and Geosmin (GSM). The regular purge and trap extraction followed by analysis with gas chromatography-mass spectrometry (GC-MS) usually involve a complicated assembly of facilities, more working space, long sample preparation time, and headspace solid-phase microextraction (SPME). In this work, a method with easier sample preparation, fewer and simplified facilities, and without SPME on GC-MS analysis is developed for the determination of 2-MIB and GSM in fish samples. Unlike previous methods, solvent extract from samples, QuEChERS-based cleanup, and solid-phase extraction for concentration are applied. The LOD (S/N > 3) and LOQ (S/N > 10) of this method were validated at 0.6 μg/kg and 1.0 μg/kg for both 2-MIB and GSM, which are under the sensory limit (1 μg/kg). Application of this method for incurred fish samples demonstrated acceptable analytical performance. This method is suitable for large-scale determination of 2-MIB and GSM in fish samples, owing to the use of simple facility and easy-to-operate procedure, rapid sample preparation, and shorter time for GC-MS analysis without SPME.

scholarly journals Water-Soluble Pyrene Tags Enable the Detection of Carbohydrates by Label-Assisted Laser Desorption/ionisation Mass Spectrometry

2020 ◽  
Author(s):  
Jacob R Hauser ◽  
Edmund T Bergström ◽  
Alexander N. Kulak ◽  
Stuart L Warriner ◽  
Jane E Thomas-Oates ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Biological Samples ◽  
Laser Desorption ◽  
Water Soluble ◽  
Maldi Ms ◽  
Ms Analysis ◽  
Ionisation Mass Spectrometry ◽  
Ionisation Mass ◽  
Reducing Carbohydrates

<div>Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) is widely used for the analysis of biomolecules. Label-assisted laser desorption/ionisation mass spectrometry (LALDI-MS) is a matrix-free variant of MALDI-MS, in which only analytes covalently attached to a laser desorption/ionisation (LDI) enhancer are detected. LALDI-MS has shown promise in overcoming limitations of MALDI-MS in terms of sample preparation and MS analysis. In this work, we have developed water-soluble pyrene-based LDI reagents (LALDI tags) that can be used for (in situ) labelling and LALDI-MS analysis of reducing carbohydrates from complex (biological) samples without the need for additional chemical derivatisation or purification. We have systematically explored the suitability of four pyrene-based LDI enhancers and three aldehyde-reactive handles, optimised sample preparation, and exemplified the use of a LALDI tag by the detection of lactose in cow’s milk. These results demonstrate that LALDI-MS is a promising technique for the analysis of reducing carbohydrates in biological samples, and pave the way for the development of LALDI-MS for glycomics and diagnostics.</div>

scholarly journals Water-Soluble Pyrene Tags Enable the Detection of Carbohydrates by Label-Assisted Laser Desorption/ionisation Mass Spectrometry

2020 ◽  
Author(s):  
Jacob R Hauser ◽  
Edmund T Bergström ◽  
Alexander N. Kulak ◽  
Stuart L Warriner ◽  
Jane E Thomas-Oates ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Biological Samples ◽  
Laser Desorption ◽  
Water Soluble ◽  
Maldi Ms ◽  
Ms Analysis ◽  
Ionisation Mass Spectrometry ◽  
Ionisation Mass ◽  
Reducing Carbohydrates

<div>Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) is widely used for the analysis of biomolecules. Label-assisted laser desorption/ionisation mass spectrometry (LALDI-MS) is a matrix-free variant of MALDI-MS, in which only analytes covalently attached to a laser desorption/ionisation (LDI) enhancer are detected. LALDI-MS has shown promise in overcoming limitations of MALDI-MS in terms of sample preparation and MS analysis. In this work, we have developed water-soluble pyrene-based LDI reagents (LALDI tags) that can be used for (in situ) labelling and LALDI-MS analysis of reducing carbohydrates from complex (biological) samples without the need for additional chemical derivatisation or purification. We have systematically explored the suitability of four pyrene-based LDI enhancers and three aldehyde-reactive handles, optimised sample preparation, and exemplified the use of a LALDI tag by the detection of lactose in cow’s milk. These results demonstrate that LALDI-MS is a promising technique for the analysis of reducing carbohydrates in biological samples, and pave the way for the development of LALDI-MS for glycomics and diagnostics.</div>

scholarly journals Optimisation of Urine Sample Preparation for Headspace-Solid Phase Microextraction Gas Chromatography-Mass Spectrometry: Altering Sample pH, Sulphuric Acid Concentration and Phase Ratio

Metabolites ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 482
Author(s):  
Prashant Aggarwal ◽  
James Baker ◽  
Mark T. Boyd ◽  
Séamus Coyle ◽  
Chris Probert ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Sample Preparation ◽  
Urine Sample ◽  
Solid Phase Microextraction ◽  
Solid Phase ◽  
Gas Chromatography Mass Spectrometry ◽  
Headspace Solid Phase Microextraction ◽  
Chromatography Mass Spectrometry ◽  
Ms Analysis

Headspace-solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) can be used to measure volatile organic compounds (VOCs) in human urine. However, there is no widely adopted standardised protocol for the preparation of urine samples for analysis resulting in an inability to compare studies reliably between laboratories. This paper investigated the effect of altering urine sample pH, volume, and vial size for optimising detection of VOCs when using HS-SPME-GC-MS. This is the first, direct comparison of H2SO4, HCl, and NaOH as treatment techniques prior to HS-SPME-GC-MS analysis. Altering urine sample pH indicates that H2SO4 is more effective at optimising detection of VOCs than HCl or NaOH. H2SO4 resulted in a significantly larger mean number of VOCs being identified per sample (on average, 33.5 VOCs to 24.3 in HCl or 12.2 in NaOH treated urine) and more unique VOCs, produced a more diverse range of classes of VOCs, and led to less HS-SPME-GC-MS degradation. We propose that adding 0.2 mL of 2.5 M H2SO4 to 1 mL of urine within a 10 mL headspace vial is the optimal sample preparation prior to HS-SPME-GC-MS analysis. We hope the use of our optimised method for urinary HS-SPME-GC-MS analysis will enhance our understanding of human disease and bolster metabolic biomarker identification.

scholarly journals Histone Sample Preparation for Bottom-Up Mass Spectrometry: A Roadmap to Informed Decisions

Proteomes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 17
Author(s):  
Simon Daled ◽  
Sander Willems ◽  
Bart Van Puyvelde ◽  
Laura Corveleyn ◽  
Sigrid Verhelst ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Research Question ◽  
Stable Gene ◽  
Assessment Procedure ◽  
Bottom Up ◽  
Genome Complexity ◽  
Arginine Residues ◽  
Multicellular Organisms ◽  
Selection Of

Histone-based chromatin organization enabled eukaryotic genome complexity. This epigenetic control mechanism allowed for the differentiation of stable gene-expression and thus the very existence of multicellular organisms. This existential role in biology makes histones one of the most complexly modified molecules in the biotic world, which makes these key regulators notoriously hard to analyze. We here provide a roadmap to enable fast and informed selection of a bottom-up mass spectrometry sample preparation protocol that matches a specific research question. We therefore propose a two-step assessment procedure: (i) visualization of the coverage that is attained for a given workflow and (ii) direct alignment between runs to assess potential pitfalls at the ion level. To illustrate the applicability, we compare four different sample preparation protocols while adding a new enzyme to the toolbox, i.e., RgpB (GingisREX®, Genovis, Lund, Sweden), an endoproteinase that selectively and efficiently cleaves at the c-terminal end of arginine residues. Raw data are available via ProteomeXchange with identifier PXD024423.

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