Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics

2006 ◽  
Vol 1 (4) ◽  
pp. 1872-1878 ◽  
Author(s):  
Brian Cox ◽  
Andrew Emili
Keyword(s):  
Mass Spectrometry ◽  
Protein Extraction ◽  
Subcellular Fractionation

Development of a C12Im-Cl-assisted method for rapid sample preparation in proteomic application

2021 ◽  
Author(s):  
Chang Liu ◽  
Xiaoxia Si ◽  
Shumei Yan ◽  
Xinyuan Zhao ◽  
Xiaohong Qian ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Protein Extraction ◽  
Sample Processing ◽  
Processing Methods ◽  
Proteomic Analyses

Chromatography and mass spectrometry (MS) techniques have greatly improved the power of proteomic analyses. However, sample processing methods, including protein extraction and digestion, before MS remain as bottlenecks in the...

Development and comparative analysis of yeast protein extraction protocols for mass spectrometry

2019 ◽  
Vol 567 ◽  
pp. 90-95 ◽  
Author(s):  
Lauana Fogaça de Almeida ◽  
Leonardo Nazário de Moraes ◽  
Lucilene Delazari dos Santos ◽  
Guilherme Targino Valente
Keyword(s):  
Mass Spectrometry ◽  
Comparative Analysis ◽  
Protein Extraction ◽  
Yeast Protein

scholarly journals A review on subcellular or organellar proteomics with special reference to apicomplexan parasites

1970 ◽  
pp. 1-7
Author(s):  
AMAM Zonaed Siddiki ◽  
MB Hossain ◽  
ASM Lutful Ahasan
Keyword(s):  
Mass Spectrometry ◽  
Special Reference ◽  
Key Words ◽  
Large Scale ◽  
Subcellular Fractionation ◽  
Apicomplexan Parasites ◽  
Proteomic Investigation ◽  
Analytical Tools ◽  
Subcellular Proteomics

Despite several well-known limitations, mass spectrometry-based proteomics is still performing important role for post-genomic investigations. As large-scale proteomic investigation of whole organism or cell has been found more complex with available analytical tools, subcellular fractionation prior to mass spectrometry is becoming more useful approach now-a-days. In this review, an attempt has been made to summarize all such subcellular or organellar proteomic investigations performed to date with its implications for apicomplexan parasites. Key words: Subcellular proteomics, parasite, mass spectrometry, fractionation DOI = 10.3329/bjvm.v5i1.1301 Bangl. J. Vet. Med. (2007). 5 (1 & 2): 01- 07

Proteomics in the Classroom: An Investigative Study of Proteins in Microorganisms

2012 ◽  
Vol 74 (4) ◽  
pp. 237-243 ◽  
Author(s):  
Jon Benskin ◽  
Sixue Chen
Keyword(s):  
Mass Spectrometry ◽  
High School ◽  
High School Students ◽  
Data Analysis ◽  
Molecular Biology ◽  
Gel Electrophoresis ◽  
Protein Extraction ◽  
Molecular Techniques ◽  
School Students

As advances in biotechnology and molecular biology rapidly expand in research settings, it is vital that we continue to prepare high school students to enter and thrive in those modern laboratories. This multistep, inquiry-based lab describes highly adaptable methods to teach students not only current molecular techniques and technologies, but also about proteomics and microorganisms. Students participate in protein extraction, gel electrophoresis, mass spectrometry, and data analysis to identify proteins present in microorganisms.

scholarly journals An Optimal Protocol to Analyze the Rat Spinal Cord Proteome

2009 ◽  
Vol 4 ◽  
pp. BMI.S2965 ◽  
Author(s):  
F. Gil-Dones ◽  
S. Alonso-Orgaz ◽  
G. Avila ◽  
T. Martin-Rojas ◽  
V. Moral-Darde ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Spinal Cord ◽  
Central Nervous System ◽  
Nervous System ◽  
Protein Separation ◽  
Protein Extraction ◽  
Total Protein Content ◽  
Cord Injury ◽  
Optimal Protocol ◽  
The Central Nervous System

Since the function of the spinal cord depends on the proteins found there, better defing the normal Spinal Cord Proteome is an important and challenging task. Although brain and cerebrospinal fluid samples from patients with different central nervous system (CNS) disorders have been studied, a thorough examination of specific spinal cord proteins and the changes induced by injury or associated to conditions such as neurodegeneration, spasticity and neuropathies has yet to be performed. In the present study, we aimed to describe total protein content in the spinal cord of healthy rats, employing different proteomics tools. Accordingly, we have developed a fast, easy, and reproducible sequential protocol for protein extraction from rat spinal cords. We employed conventional two dimensional electrophoresis (2DE) in different pH ranges (eg. 4–7, 3–11 NL) combined with identification by mass spectrometry (MALDI-TOF/TOF), as well as first dimension protein separation combined with Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LC-MS/MS) to maximise the benefits of this technology. The value of these techniques is demonstrated here by the identification of several proteins known to be associated with neuroglial structures, neurotransmission, cell survival and nerve growth in the central nervous system. Furthermore this study identified many spinal proteins that have not previously been described in the literature and which may play an important role as either sensitive biomarkers of dysfunction or of recovery after Spinal Cord Injury.

scholarly journals Gel Electrophoresis/Electroelution Sorting Fractionator combined with Filter Aided Sample preparation (FASP) for deep proteomic analysis

2021 ◽  
Author(s):  
Yassel Ramos ◽  
Alexis Almeida ◽  
Jenis Carpio ◽  
Arielis Rodríguez-Ulloa ◽  
Yasser Perera ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Protein Identification ◽  
Protein Extraction ◽  
Complex Mixture ◽  
Fold Increase ◽  
Mass Spectrometry Analysis ◽  
Protein Fractionation ◽  
Spectrometry Analysis ◽  
Sds Page

AbstractSample preparation and protein fractionation are important issues in proteomic studies in spite of the technological achievements on protein mass spectrometry. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is SDS-PAGE-based protein fractionation due to its extraordinary resolution and the effectiveness of SDS as a solubilizing agent. Its main limitation lies in the poor recovery of the gel-trapped proteins, where protein electro-elution is the most successful approach to overcome this drawback. We created a device to separate complex mixture of proteins and peptides (named “GEES fractionator”) that is based on the continuous Gel Electrophoresis/Electro-elution Sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electro-eluted to the solution containing wells. The performance of the device was studied for SDS-PAGE-based protein fractionation in terms of reproducibility, protein recovery and loading capacity. In the SDS-free PAGE setup, complex peptide mixtures can also be fractionated. More than 11 700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the GEES fractionator combined with the Filter Aided Sample Preparation (FASP) method and mass spectrometry analysis. GEES-based proteome characterization shows a 1.7 fold increase in the number of identified proteins compared to the unfractionated sample analysis. Proteins involved in the co-regulated transcription activity, as well as cancer related pathways such as apoptosis signaling, P53 and RAS pathways are more represented in the protein identification output of GEES-based fractionation approaches.

scholarly journals Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane

2004 ◽  
Vol 167 (6) ◽  
pp. 1099-1112 ◽  
Author(s):  
Marcello Marelli ◽  
Jennifer J. Smith ◽  
Sunhee Jung ◽  
Eugene Yi ◽  
Alexey I. Nesvizhskii ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Large Scale ◽  
Secretory Pathway ◽  
Subcellular Fractionation ◽  
Membrane Dynamics ◽  
Small Gtpase ◽  
Quantitative Mass Spectrometry ◽  
Actin Organization ◽  
Associated Proteins ◽  
Peroxisome Membrane

We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.

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