Breaking News
December 14, 2018 - Scientists perform lung lavage as new approach for tuberculosis diagnosis in rhinoceros
December 14, 2018 - Recent winners of the Nobel Medicine Prize
December 14, 2018 - KHN’s ‘What the Health?’ Insurance enrollment is lagging — and there are lots of reasons why
December 14, 2018 - Study assesses safety and efficacy of new treatment for pancreatic cancer
December 14, 2018 - Study finds drug targets for Ebola, Dengue, and Zika viruses
December 14, 2018 - Study highlights need for personalized approach to treat ICU acquired delirium
December 14, 2018 - Massage helps relieve pain, improve mobility in patients with knee osteoarthritis
December 14, 2018 - Average outpatient visit in the U.S. costs nearly $500, shows new study
December 14, 2018 - Reference Infliximab, Biosimilar Equivalent for Crohn’s Disease
December 14, 2018 - New contact lens to treat eye injuries
December 14, 2018 - Acne could have a genetic basis find researchers promising new cure
December 14, 2018 - Higher physical activity associated with improved mood
December 14, 2018 - New UGA study points to optimal hypertension treatment for stroke patients
December 14, 2018 - Study highlights factors that can reduce food cravings
December 14, 2018 - Researchers discover Ebola-fighting protein in human cells
December 14, 2018 - Fentanyl surpasses heroin in cause of U.S. drug overdose deaths
December 14, 2018 - When Heart Attack Strikes, Women Often Hesitate to Call for Help
December 14, 2018 - A warning about costume contacts
December 14, 2018 - Study examines link between peripheral artery disease and heart attack
December 14, 2018 - Researchers develop biotechnological tool to produce antifungal proteins in plants
December 14, 2018 - 3D-printed adaptive aids can benefit patients with arthritis
December 14, 2018 - Chronic bullying during adolescence impacts mental health
December 14, 2018 - Integral Molecular and Merus collaborate to develop bispecific antibody therapeutics
December 13, 2018 - Importance of cell cycle and cellular senescence in the placenta discovered
December 13, 2018 - Gold “nanoprisms” open new window into vessels and single cells
December 13, 2018 - Research findings could lead to new targets for cancer-fighting therapeutics
December 13, 2018 - Butantan Institute signs collaboration agreement with MSD to develop dengue vaccines
December 13, 2018 - Study explores how patients want to discuss symptoms with doctors
December 13, 2018 - RUDN medics first to gather scattered data on hepatitis morbidity in Somalia
December 13, 2018 - Age and gender disparities found in use of bed nets to prevent malaria in sub-Saharan Africa
December 13, 2018 - Caffeine therapy benefits developing brains of premature babies
December 13, 2018 - New review focuses on electrospinning techniques used in musculoskeletal tissue engineering
December 13, 2018 - A new division focused on human immune system
December 13, 2018 - Zogenix Announces Positive Phase 3 Trial Results on the Efficacy and Safety of Fintepla (ZX008) in Dravet Syndrome
December 13, 2018 - BCR ABL Genetic Test: MedlinePlus Lab Test Information
December 13, 2018 - Caffeinated beverages during pregnancy linked to lower birth weight babies
December 13, 2018 - Stanford Medicine Health Trends Report examines opportunity to democratize health care
December 13, 2018 - Obsessive-compulsive disorder may protect individuals from obesity
December 13, 2018 - Scientists investigate how a painful event is processed in the brain
December 13, 2018 - Genetic study reveals new insights into underlying causes of moderate-to-severe asthma
December 13, 2018 - Study uncovers new genetic clues to frontotemporal dementia
December 13, 2018 - Vitamin C supplementation for pregnant smokers may reduce harm to infants’ lungs
December 13, 2018 - New study reveals yin-yang personality of dopamine
December 13, 2018 - Research identifies nerve-signaling pathway behind sustained pain after injury
December 13, 2018 - Children with high levels of callous traits show widespread differences in brain structure
December 13, 2018 - Long-term Benefit of Steroid Injections for Knee Osteoarthritis Challenged
December 13, 2018 - Adding new channels to the brain remote control
December 13, 2018 - In the Spotlight: A different side of neuroscience
December 13, 2018 - Medical Marvels: Using immunotherapy for melanoma that spread to the brain
December 13, 2018 - Puzzles do not keep dementia away finds study
December 13, 2018 - New mouse model shows potential for rapid identification of promising muscular dystrophy therapies
December 13, 2018 - Study reveals urban and rural differences in prenatal exposure to essential and toxic elements
December 13, 2018 - New collaborative partnership in quest of novel antibiotics
December 13, 2018 - Single tau molecule holds clues to help diagnose neurodegeneration in its earliest stages
December 13, 2018 - AHA Scientific Statement: Low Risk of Side Effects for Statins
December 13, 2018 - What Is Acute Flaccid Myelitis?
December 13, 2018 - How bereaved people control their thoughts without knowing it
December 13, 2018 - Health care democratization underway, according to 2nd annual Stanford Medicine Health Trends Report | News Center
December 13, 2018 - Going Beyond a Single Color
December 13, 2018 - London-based startup launches ‘thedrug.store’ aiming to clean up CBD industry
December 13, 2018 - Loss of tight junction barrier protein results in gastric cancer development
December 13, 2018 - Novel way to efficiently deliver anti-parasitic medicines
December 13, 2018 - RKI publishes new data on disease prevention and utilization of medical services
December 13, 2018 - High-tech, flexible patches sewn into clothes could help to stay warm
December 13, 2018 - The CCA releases three reports on requests for medical assistance in dying
December 13, 2018 - Restoring Hair Growth on Scarred Skin? Mouse Study Could Show the Way
December 13, 2018 - Probiotic use may reduce antibiotic prescriptions, researchers say
December 13, 2018 - Drug repositioning strategy identifies potential new treatments for epilepsy
December 13, 2018 - Chronic rhinitis associated with hospital readmissions for asthma and COPD patients
December 13, 2018 - Food poisoning discovery could save lives
December 13, 2018 - Cloned antibodies show potential to treat, diagnose life-threatening fungal infections
December 13, 2018 - Exercise may reduce colorectal cancer risk after weight loss
December 13, 2018 - Russian scientists create hardware-information system for brain disorders treatment
December 13, 2018 - Moderate alcohol consumption linked with lower risk of hospitalization
December 13, 2018 - Nurturing Healthy Neighborhoods | NIH News in Health
December 13, 2018 - Rise in meth and opioid use during pregnancy
December 13, 2018 - Researchers gain new insights into pediatric tumors
December 13, 2018 - FSU study finds racial disparity among adolescents receiving flu vaccine
December 13, 2018 - Study investigates attitudes toward implementation of ‘sex as a biological variable’ policy
December 13, 2018 - Drug cocktail induces cancer cell death by switching off energy supply
Researchers optimize method to increase resolution of proteomic analysis

Researchers optimize method to increase resolution of proteomic analysis

image_pdfDownload PDFimage_print

One of the key challenges of proteomics, the study of all proteins expressed by a cell or organism, is managing to distinguish between molecules that are structurally different yet have the same mass.

This is hard because a mass spectrometer, the main apparatus used in this type of study, works like a weighing scale, sorting the molecules analyzed according to their mass.

One way to reduce confusion when using a mass spectrometer is to start by submitting the sample to liquid chromatography, which separates hydrophilic (“water-loving”) proteins from hydrophobic ones. The hydrophilic proteins enter the spectrometer first, and the most hydrophobic are left for the last, decreasing the likelihood that two different molecules with equivalent masses will be interpreted as only one by the apparatus.

“It’s like solving a jigsaw puzzle with millions of pieces. When you first open the bag, the pieces are all jumbled and overlapping. You must begin by sorting them out. As we work with proteomics, we constantly endeavor to develop more refined sorting techniques,” said Daniel Martins-de-Souza, who heads the Neuroproteomics Laboratory at the University of Campinas (UNICAMP) in Brazil.

In a study with results recently published in Proteomics and featured on the cover of the journal, Martins-de-Souza’s group optimized a method to increase the resolution of proteomic analysis by mass spectrometry. Thanks to a combination of two other techniques – two-dimensional liquid chromatography and ion mobility – the group succeeded in identifying 10,390 proteins expressed in oligodendrocytes, the central nervous system cells responsible for producing myelin, a lipidic substance that plays an essential role in the information exchange between neurons.

With FAPESP’s support, the UNICAMP group have studied the human oligodendrocyte proteome for several years, with the aim of better understanding the causes of schizophrenia as a basis for proposing novel therapeutic approaches.

“We now have a far more complete oligodendrocyte protein database, which will be useful for our own studies and those of other researchers in the field,” Martins-de-Souza said. “It’s available online, and the data can be downloaded. In addition, the optimization technique can be used to study the proteome of any biological sample.”

In a previous study using single-dimensional liquid chromatography for pre-sorting, the group had identified only 2,290 proteins in oligodendrocytes.

Technology application

According to Martins-de-Souza, currently available treatments for schizophrenia focus on neurons, but the neural communication failures observed in patients may be due to oligodendrocyte dysfunction.

“One of our research lines consists of evaluating how the drugs used to control schizophrenia modify the oligodendrocyte proteome,” he said. “With this new methodology, we can obtain five times more information on the role of these drugs.”

The study was conducted during the postdoctoral research of Juliana Silva Cassoli and the master’s research of Caroline Brandão Teles, both with scholarships from FAPESP and supervision by Martins-de-Souza.

How it works

The first step in proteomic analysis using mass spectrometry is to break down the proteins extracted from the biological sample of interest, which in this case consists of oligodendrocytes, into smaller particles called peptides.

“A small protein can give rise to at least ten different peptides. The spectrometer isn’t good at analyzing the entire molecule because of its large size,” Martins-de-Souza explained.

Next the group submitted the sample to separation by chromatography. Instead of using a single matrix, as in the conventional technique, they used two. In the first separation, only a fifth of the injected peptides entered the spectrometer in liquid form. This was followed by another fifth in the second separation, and so on.

“It’s as if you spread out the pieces of the jigsaw puzzle with both hands instead of only one,” Martins-de-Souza said.

Inside the spectrometer, the sample is transformed into gas and flies back and forth in a vacuum. The smaller the peptide, the faster it reaches its destination, and the apparatus then measures its mass.

As the molecules are flying about inside the spectrometer, the ion mobility technique injects a small amount of gas into the apparatus through a tube.

“The resistance offered to the gas by the molecule depends on its three-dimensional shape, so if two different peptides with the same mass are flying together and we inject the gas in the opposite direction, they will tend to be separated by the force of resistance to the gas. It’s like picking up two sheets of paper with the same mass, crumpling one into a ball, and dropping them both. Because of its shape, the crumpled sheet will reach the floor first,” Martins-de-Souza explained.

At the end of the experiment, the more than 223,000 peptides identified by the spectrometer were reconstructed using bioinformatics tools, resulting in the 10,390 proteins described in the paper. The group also used bioinformatics to map the cellular compartments in which the proteins are located and the biological processes in which they are involved.

“Ideally, it should be possible to identify at least two peptides per protein. That way, we can be sure a molecule is really present in the sample, since two proteins with two exactly identical peptides are unlikely to occur. In this study, about 20% of the proteins were identified by more than 20 peptides,” Martins-de-Souza said.

The methodology enabled the researchers to identify even proteins that were relatively scarce in the sample, i.e., in quantities some 10 million times smaller than those of the most highly expressed molecules.

“One of the problems with mass spectrometry is that a very large piece of the jigsaw puzzle may hide several smaller ones. However, with an effective tool to spread out the pieces, you can see practically all of them,” Martins-de-Souza said.

Source:

http://agencia.fapesp.br/novel_methodology_increases_resolution_in_oligodendrocyte_proteomics/26932/

Tagged with:

About author

Related Articles