scholarly journals Assessment and refinement of sample preparation methods for deep and quantitative plant proteome profiling

2018 ◽  
Author(s):  
Gaoyuan Song ◽  
Polly Y Yingshan Hsu ◽  
Justin W. Walley
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Protein Extraction ◽  
Leaf Tissue ◽  
Extraction Methods ◽  
Label Free ◽  
Preparation Methods ◽  
Proteome Profiling ◽  
Leaf Tissues ◽  
Sample Preparation Methods

SummaryA major challenge in the field of proteomics is obtaining high quality peptides for comprehensive proteome profiling by liquid chromatography mass spectrometry for many organisms. Here we evaluate and modify a range of sample preparation methods using photosynthetically active Arabidopsis leaf tissues from several developmental timepoints. We find that inclusion of FASP-based on filter digestion improves all protein extraction methods tested. Ultimately, we show that a detergent-free urea-FASP approach enables deep and robust quantification of leaf proteomes. For example, from 4-day-old leaf tissue we profiled up to 11,690 proteins from a single sample replicate. This method should be broadly applicable to researchers working on difficult to process samples from a range of plant and non-plant organisms.AbbreviationsChloroMethanol/Chloroform ExtractionFASPFilter Aided Sample PrepGOGene OntologyIAAIodoacetamideLFQLabel Free QuantificationMS/MSTandem mass spectrometryTFTranscription FactorUAUrea Extraction1D1 Dimensional2D2 Dimensional

scholarly journals Evaluation of sample preparation methods for mass spectrometry-based proteomic analysis of barley leaves

Plant Methods ◽  
2018 ◽  
Vol 14 (1) ◽  
Author(s):  
Wei-Qing Wang ◽  
Ole Nørregaard Jensen ◽  
Ian Max Møller ◽  
Kim H. Hebelstrup ◽  
Adelina Rogowska-Wrzesinska
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Proteomic Analysis ◽  
Preparation Methods ◽  
Barley Leaves ◽  
Sample Preparation Methods

scholarly journals Comparison of methods to examine the endogenous peptides of fetal calf serum

2021 ◽  
Author(s):  
Declan Williams ◽  
Peihong Zhu ◽  
Peter Bowden ◽  
Catherine Stacey ◽  
Mike McDonell ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Sample Preparation ◽  
Tandem Mass Spectrometry ◽  
Amino Acid Substitutions ◽  
Tandem Mass ◽  
Preparation Methods ◽  
Endogenous Peptides ◽  
Maldi Tof ◽  
Sample Preparation Methods

There is a great desire to relate the patterns of endogenous peptides in blood to human disease and drug response. The best practices for the preparation of blood fluids for analysis are not clear and also relatively few of the peptides in blood have been identified by tandem mass spectrometry. We compared a number of sample preparation methods to extract endogenous peptides including C18 reversed phase, precipitation, and ultrafiltration. We examined the results of these sample preparation methods by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-tandem mass spectrometry (MS/MS) using MALDI-TOF/TOF and electrospray ionization-ion trap. Peptides from solid-phase extraction with C18 in the range of hundreds of femtomoles per spot were detected from the equivalent of 1 μL of serum by MALDI-TOF. We observed endogenous serum peptides from fibrinogen α- and β-chain, complement C3, α-2-HS-glycoprotein, albumin, serine (or cysteine) proteinase inhibitor, factor VIII, plasminogen, immunoglobulin, and other abundant blood proteins. However, we also recorded significant MS/MS spectra from tumor necrosis factor-α-, major histocompatibility complex-, and angiotensin-related peptides, as well as peptides from collagens and other low-abundance proteins. Amino acid substitutions were detected and a phosphorylated peptide was also observed. This is the first time the endogenous peptides of fetal serum have been examined by MS and where peptides from low-abundance proteins, phosphopeptides, and amino acid substitutions were detected.

scholarly journals Phytochelatin Characterization in Commonly Consumed Plant Foods Using Mass Spectrometry-based Metabolomics (P18-122-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Kristine Dennis ◽  
Ken Liu ◽  
ViLinh Tran ◽  
Bill Liang ◽  
Young-Mi Go ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Detection Methods ◽  
Fragmentation Pattern ◽  
Human Diet ◽  
Preparation Methods ◽  
Plant Foods ◽  
Ion Dissociation ◽  
Fragmentation Patterns ◽  
Sample Preparation Methods

Abstract Objectives Phytochelatins (PyCs) are a group of metal-binding compounds formed by plants which could impact bioavailability of essential and toxic metals in the human diet. Liquid chromatography mass spectrometry (LC-MS)-based metabolomics can characterize a diversity of compounds from complex matrices. The aim of this project was to determine chromatographic characteristics of PyCs and their mass spectral signatures using authentic standards and determine if two types of PyCs (PyC2-Gly and PyC3-Gly) can be detected in commonly consumed plant foods and validated using chromatographic characteristics and ion dissociation mass spectrometry (MS/MS). Methods We analyzed PyC2-Gly and PyC3-Gly standards using LC-MS (C18 column; 10 minute analysis, positive ionization mode) at 5 different concentrations with 3 sample preparation methods to determine the common adducts, retention times, and optimal sample preparation methods. The most common adducts were selected for MS/MS to determine the characteristic fragmentation patterns. Onion, carrot, tomato, and whole wheat flour samples were analyzed with LC-MS/MS using the optimized sample preparation method and determined chromatographic and ion dissociation characteristics from the PyC standards analysis. Results For both PyC standards, the most common adducts were identified as follows: M + H > M + Na > M +K > 2M + H > 2M + Na = 2M + K > M + 2H and metabolite features had a retention time of 42 seconds. The fragmentation patterns for PyC2-Gly and PyC3-Gly for the M + H adduct (m/z 538.1272 and m/z 770.1790) were determined by MS/MS. The results show that fragmentation of m/z 538.1272 yielded m/z 231.0433, 334.0525, and 409.0844, and m/z 770.1790 yielded m/z 231.0433, 308.0911, and 641.1358. PyC2-Gly was detected in all 4 food types with a fragmentation pattern matching the PyC2-Gly standard while PyC3-Gly was not detected in these food samples. Conclusions This project defines chromatographic and mass spectrometry characteristics of PyC2-Gly and PyC3-Gly. Using these characteristics, we were able to validate PyC2-Gly in 4 plant foods. As PyCs may impact metal bioavailability from the diet, optimizing LC-MS detection methods for PyCs in plant foods facilitates future characterization of PyCs in the human diet. Funding Sources National Institute of Environmental Health Sciences.

Recent technical progress in sample preparation and liquid-phase separation-mass spectrometry for proteomic analysis of mass-limited samples

2021 ◽  
Vol 13 (10) ◽  
pp. 1214-1225
Author(s):  
Zhichang Yang ◽  
Liangliang Sun
Keyword(s):  
Mass Spectrometry ◽  
Phase Separation ◽  
Liquid Phase ◽  
Sample Preparation ◽  
Technical Progress ◽  
Proteomic Analysis ◽  
Liquid Phase Separation ◽  
Preparation Methods ◽  
Sample Preparation Methods

Recent development of sample preparation methods with nanoliter processing volumes, ultrasensitive LC-MS/MS and CZE-MS/MS systems for proteomics of mass-limited samples.

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