article Flashcards
What is Proteomics
• Proteomics is the science and technology of separating and identifying proteins from crude biological samples.
When is Proteomics significant and what will this allow for
It is mostly significant when differentially-expressed proteins between two samples that belong
or that are subject to different conditions are identified
This identification will allow for the characterization of biological roles, clarification of biological mechanisms, and identification of therapeutic targets and biomarkers. .
What is the first step in proteomic study
The first step of a proteomic study requires almost always the separation of proteins using electrophoresis or chromatography techniques followed by the identification steps that are performed using mass spectrometry.
what does mass spectrometry measure and what is the identification based on
The latter measures accurately the masses of peptides generated from the digestion of the protein by trypsin or another enzyme followed by the use of software that scan the different protein databases available to identify the protein.
This identification is based on the information collected about this protein throughout the study including but not limited to isoelectric point, molecular mass, peptide masses, and specie of the biological sample. It is preferable–even required sometimes–to use immunoblotting techniques or N-terminal sequencing to validate the identity of the proteins.
Proteomics studies can be applied to what
Proteomics studies can be applied on any protein mixture extracted from any organism including plant, bacterial, and animal cells.
Proteomics strategies have been used to identify what
Proteomics strategies have been used to identify disease-specific protein markers called biomarkers that could provide the basis for the development of new diagnosis methodologies, treatments, and early disease detection
describe whole process of Proteomics
• It is mostly significant when differentially-expressed proteins between two samples that belong
or that are subject to different conditions are identified.
• This identification will allow for the characterization of biological roles, clarification of biological mechanisms, and identification of therapeutic targets and biomarkers.
• The first step of a proteomic study requires almost always the separation of proteins using electrophoresis or chromatography techniques followed by the identification steps that are performed using mass spectrometry.
• The latter measures accurately the masses of peptides generated from the digestion of the protein by trypsin or another enzyme followed by the use of software that scan the different protein databases available to identify the protein.
• This identification is based on the information collected about this protein throughout the study including but not limited to isoelectric point, molecular mass, peptide masses, and specie of the biological sample. It is preferable–even required sometimes–to use immunoblotting techniques or N-terminal sequencing to validate the identity of the proteins.
• Proteomics studies can be applied on any protein mixture extracted from any organism including plant, bacterial, and animal cells.
• Proteomics strategies have been used to identify disease-specific protein markers called biomarkers that could provide the basis for the development of new diagnosis methodologies, treatments, and early disease detection
what does clinical proteomics mean
• The term clinical proteomics refers to “the application of available proteomics technologies to current areas of clinical investigation”
how do Many diseases manifest themselves and what does it form the basis of
Many diseases manifest themselves through severe changes in human physiology, which forms the basis for clinical chemistry and presents its value in diagnoses and subsequent therapeutic interventions
what does Clinical proteomics include and whats its aim
Clinical proteomics includes the global analysis of proteins expressed by the genome of an organism, with the typical aim being the evaluation of quantitative changes that occur as a function of disease, treatment, or environment
define biomarker
A biomarker is defined as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathological processes, or pharmacological responses to a therapeutic intervention
how are biomarkers judged by their effectivness
• The degree to which biomarkers reflect clinical outcomes judges their effectiveness.
As biomarkers have different characteristics there should be what requirements and what does it determine
As biomarkers have different characteristics there should be statistical requirements that determine the usefulness of biomarkers as evaluations of disease progression or outcomes in clinical trials
what should biomarkers requirements include
Those requirements should include
- statistical dispersion
- detailed information on target populations,
- specificity of the biomarker.
what is an ideal biomarker expected to me able to do - 3things
Generally, an ideal biomarker is expected to be: able to
- detect a fundamental feature of a specific disease; validated in and confirmed by those specific disease cases;
- precise,
- able to detect the early stages of this specific disease 4. and distinguish it from other similar disease cases or family members of that disease;
- simple to perform;
- reliable;
- non-invasive;
- inexpensive if possible
what does (MudPIT) stand for
implementation of multi-dimensional protein identification technology
To identify new biomarkers of a certain disease in vitro, a what was developed , which consists of what
three component analytical strategy
(I) a cell line as the pre-clinical model,
(II) a set of five well-studied drugs, three of which had been found in humans to elicit that disease, and
(III) implementation of multi-dimensional protein identification technology (MudPIT) to perform semiquantitative analysis and identify protein biomarker candidates
why was MudPIT was chosen
due to the demonstration of its usefulness in the identification as well as semi-quantification (relative changes/differential trends in protein abundance) of large numbers of proteins, both in vivo and in vitro
This biomarker-identification stage generates what
• This biomarker-identification stage generates a large list of biomarkers.
what is the second stage of biomarkers called and what happens when it passes it
• Upon passing through a crucial second stage, a rate determining one, only the most appropriate subset of biomarker-candidates will be further tested by traditional immunoassays to identify and verify an ideal biomarker or the most credible biomarker for a specific disease according to aforementioned criteria
describe all of 1.2 Clinical proteomics and biomarkers
- The term clinical proteomics refers to “the application of available proteomics technologies to current areas of clinical investigation”
- Many diseases manifest themselves through severe changes in human physiology, which forms the basis for clinical chemistry and presents its value in diagnoses and subsequent therapeutic interventions
- Clinical proteomics includes the global analysis of proteins expressed by the genome of an organism, with the typical aim being the evaluation of quantitative changes that occur as a function of disease, treatment, or environment
- A biomarker is defined as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathological processes, or pharmacological responses to a therapeutic intervention”
- The degree to which biomarkers reflect clinical outcomes judges their effectiveness.
- As biomarkers have different characteristics there should be statistical requirements that determine the usefulness of biomarkers as evaluations of disease progression or outcomes in clinical trials
- Those requirements should include statistical dispersion, detailed information on target populations, and specificity of the biomarker.
- Generally, an ideal biomarker is expected to be: able to detect a fundamental feature of a specific disease; validated in and confirmed by those specific disease cases; precise, able to detect the early stages of this specific disease and distinguish it from other similar disease cases or family members of that disease; simple to perform; reliable; non-invasive; and inexpensive if possible
- To identify new biomarkers of a certain disease in vitro, a three component analytical strategy was developed, which consists of (I) a cell line as the pre-clinical model, (II) a set of five well-studied drugs, three of which had been found in humans to elicit that disease, and (III) implementation of multi-dimensional protein identification technology (MudPIT) to perform semiquantitative analysis and identify protein biomarker candidates
- MudPIT was chosen due to the demonstration of its usefulness in the identification as well as semi-quantification (relative changes/differential trends in protein abundance) of large numbers of proteins, both in vivo and in vitro
- This biomarker-identification stage generates a large list of biomarkers.
- Upon passing through a crucial second stage, a rate determining one, only the most appropriate subset of biomarker-candidates will be further tested by traditional immunoassays to identify and verify an ideal biomarker or the most credible biomarker for a specific disease according to aforementioned criteria
What is the most most studied species in terms of protein profiling studies and this species cell line have been used for what
• Mammalian samples are the most studied species in terms of protein profiling studies. In vitro culture of mammalian cell lines is an important resource for research, and have been used for disease-related studies as well as technology related ones
The protein profiles of cancerous cell lines are compared to what
the profiles of normal cell lines
While tumor-derived cell lines benefits and disadvantage
While tumor-derived cell lines can be useful for initial studies, each line displays a unique evolution that may not truly mimic real in vivo conditions
A comparison between human prostate cell lines with tumor cells from prostate patients showed what
significant altered protein profiles
Differentially-expressed proteins identified in human in vivo cancerous tissues when compared to their normal counterparts are by far more significant than what
than in vitro ones.
what have a large number of studies been sucessful in
A large number of studies have been successful in identifying protein signatures of a disease or a condition from in vivo tissues as well as patient serum samples
These differentially-expressed proteins are considered what in an organism
These differentially-expressed proteins are considered either the cause or the effect of the physiological change in the organism.
Many published proteomic studies of human tumor tissue are associated with what and what do the stduies incllude
- Many published proteomic studies of human tumor tissue are associated with weaknesses in tumor representation, sample contamination by nontumor cells and serum proteins.
- Studies often include a moderate number of tumors which may not be representative of clinical materials
1.3 Significance of proteomics of human samples
- Mammalian samples are the most studied species in terms of protein profiling studies. In vitro culture of mammalian cell lines is an important resource for research, and have been used for disease-related studies as well as technology related ones
- The protein profiles of cancerous cell lines have been compared to the profiles of normal cell lines
- While tumor-derived cell lines can be useful for initial studies, each line displays a unique evolution that may not truly mimic real in vivo conditions
- A comparison between human prostate cell lines with tumor cells from prostate patients showed significant altered protein profiles
- Differentially-expressed proteins identified in human in vivo cancerous tissues when compared to their normal counterparts are by far more significant than in vitro ones.
- A large number of studies have been successful in identifying protein signatures of a disease or a condition from in vivo tissues as well as patient serum samples
- These differentially-expressed proteins are considered either the cause or the effect of the physiological change in the organism.
- Many published proteomic studies of human tumor tissue are associated with weaknesses in tumor representation, sample contamination by nontumor cells and serum proteins.
- Studies often include a moderate number of tumors which may not be representative of clinical materials
What is human serum
- Human serum is the clear portion of the human’s body fluid that separates from blood upon clotting.
- This clear fluid provides moisture to the serous membranes in the human body.
- Normal human serum mainly contains the following proteins: IgG, IgM (Ekdahl et al. 1994), IgA, haptoglobulin, albumin, transferrin , α1-anti-trypsin, fi brinogen, α2-Macroglobulin, and complement C3, those account for >95% of total serum proteins, many of which are synthesized and secreted, shed, or lost from cells and tissues throughout the body
function of clear fluid
This clear fluid provides moisture to the serous membranes in the human body.
serum contains how much proteins? in addition to various small moleclue ssuch as what?
It contains 60–80 mg/mL of proteins in addition to various small molecules including amino acids, lipids, salts, and sugars
Normal human serum mainly contains the following what proteins:
Normal human serum mainly contains the following proteins: IgG, IgM (Ekdahl et al. 1994), IgA, haptoglobulin, albumin, transferrin , α1-anti-trypsin, fi brinogen, α2-Macroglobulin, and complement C3, those account for >95% of total serum proteins, many of which are synthesized and secreted, shed, or lost from cells and tissues throughout the body
Analysis of the human serum proteome, especially for biomarkers has potetinal for what
• Analysis of the human serum proteome, especially for biomarkers, has great potential for diagnosis and early detection of human disease
One of the difficulties to identifying a specific marker in the human serum is what
One of the difficulties to identifying a specific marker in the human serum is the low abundance of proteins secreted in the serum as a result of the disease when compared to the high concentration of albumin, resulting from daily synthesis of~12 g in the liver and a half-life of about 21 days , that constitutes more than 50% (w/w) of the total amount of proteins in the serum.
Knowing the complexity of the human proteome and the broad dynamic range in abundance of individual proteins is a need for what
and what step is needed to eliminate somethinf from serum
Knowing the complexity of the human proteome and the broad dynamic range in abundance of individual proteins (e.g. albumin, immunoglobulin), there is a need for sample treatment prior to biomarker identification and is feasible using different analytical techniques.
• A prefractionation step to eliminate albumin from the serum is therefore required prior to the proteomic study
1.4 Human serum
- Human serum is the clear portion of the human’s body fluid that separates from blood upon clotting.
- This clear fluid provides moisture to the serous membranes in the human body. It contains 60–80 mg/mL of proteins in addition to various small molecules including amino acids, lipids, salts, and sugars
- Normal human serum mainly contains the following proteins: IgG, IgM (Ekdahl et al. 1994), IgA, haptoglobulin, albumin, transferrin , α1-anti-trypsin, fi brinogen, α2-Macroglobulin, and complement C3, those account for >95% of total serum proteins, many of which are synthesized and secreted, shed, or lost from cells and tissues throughout the body
- Analysis of the human serum proteome, especially for biomarkers, has great potential for diagnosis and early detection of human disease
- One of the difficulties to identifying a specific marker in the human serum is the low abundance of proteins secreted in the serum as a result of the disease when compared to the high concentration of albumin, resulting from daily synthesis of~12 g in the liver and a half-life of about 21 days , that constitutes more than 50% (w/w) of the total amount of proteins in the serum.
- Knowing the complexity of the human proteome and the broad dynamic range in abundance of individual proteins (e.g. albumin, immunoglobulin), there is a need for sample treatment prior to biomarker identification and is feasible using different analytical techniques.
- A prefractionation step to eliminate albumin from the serum is therefore required prior to the proteomic study
Proteins are composed of what
- Proteins are composed of covalently bound amino acids.
- There are 20 different amino acids generating an infinite number of possible proteins.
- Several protein physical properties should be taken into consideration for a successful proteomic project.
- These properties are used to separate and to identify proteins from crude biological samples.
- Based on the side chain properties, amino acids of a protein can form unstable interactions through electrostatic, hydrogen bonding, and hydrophobic affinities.
- These interactions result in the folding of proteins that hinders their separation and identification necessitating the introduction of several reagents to the protein solubilizing buffer to break these interactions.
- Once these interactions are neutralized, the protein becomes unfolded (denatured).
- After denaturing the proteins, the remaining physical properties are used to separate and purify these proteins
how many amino acids are there to genertae lots of different possible proteins
There are 20 different amino acids generating an infinite number of possible proteins.
in proteins what should be taken into account for a successful proteomic project.
Several protein physical properties should be taken into consideration for a successful proteomic project
protein properties are used for what
These properties are used to separate and to identify proteins from crude biological samples
Based on the side chain properties, amino acids of a protein can form unstable interactions through what and what do these interactions result in
Based on the side chain properties, amino acids of a protein can form unstable interactions through electrostatic, hydrogen bonding, and hydrophobic affinities.
These interactions result in the folding of proteins that hinders their separation and identification necessitating the introduction of several reagents to the protein solubilizing buffer to break these interactions.
what happens after protein interactions are neutralized
Once these interactions are neutralized, the protein becomes unfolded (denatured).
•
what happens after denaturing of proteins
After denaturing the proteins, the remaining physical properties are used to separate and purify these proteins
proteins
- Proteins are composed of covalently bound amino acids.
- There are 20 different amino acids generating an infinite number of possible proteins.
- Several protein physical properties should be taken into consideration for a successful proteomic project.
- These properties are used to separate and to identify proteins from crude biological samples.
- Based on the side chain properties, amino acids of a protein can form unstable interactions through electrostatic, hydrogen bonding, and hydrophobic affinities.
- These interactions result in the folding of proteins that hinders their separation and identification necessitating the introduction of several reagents to the protein solubilizing buffer to break these interactions.
- Once these interactions are neutralized, the protein becomes unfolded (denatured).
- After denaturing the proteins, the remaining physical properties are used to separate and purify these proteins
Proteins are amphoteric molecules: meaning what
1.5.1 Isoelectric point (pI)
• Proteins are amphoteric molecules: they can carry positive, negative or a neutral charge.
At a certain pH of proteins what happens
At a certain pH, the number of positive charges is equal to the number of negative charges: this pH is equal to the pI of the protein.
The amino acids that affect the value of the protein isoelectric point the most are what
The amino acids that affect the value of the protein isoelectric point the most are those with ionizable side chains: Arginine, Tyrosine, Lysine, Cysteine, Histidine, Glutamic acid, and Aspartic acid.
Proteins can be separated based on what and using what
Proteins can be separated based on their isolelectric point using ion exchange chromatography as well as immobilized pH gradient polyacrylamide gels
highest hydrophobic side chains are what
1.5.2 Hydrophobicity
- The highly hydrophobic amino acid side chains are those of: Valine, Leucine, Isoleucine, Methionine, Phenylalanine, Tryptophan, and Cysteine.
- Proteins can be separated based on their hydrophobicities using Reversed-Phased High performance Liquid Chromatography (RP-HPLC).
- The stationary phase used for RP-HPLC is composed of silica beads with carbon chains linked to it.
- The protein mixture is injected in the column and then an acetonitrile gradient is imposed.
- More hydrophobic proteins require higher concentrations of acetonitrile for their elution
wha does (RP-HPLC). stand for
Reversed-Phased High performance Liquid Chromatography (RP-HPLC).
Proteins can be separated based on their hydrophobicities using what
Proteins can be separated based on their hydrophobicities using Reversed-Phased High performance Liquid Chromatography (RP-HPLC).
The stationary phase used for RP-HPLC is composed of what
and what is the protein mixture injected into to
The stationary phase used for RP-HPLC is composed of silica beads with carbon chains linked to it.
The protein mixture is injected in the column and then an acetonitrile gradient is imposed.
More hydrophobic proteins require higher concentrations of what for what
More hydrophobic proteins require higher concentrations of acetonitrile for their elution
Most of the time, denatured proteins having a higher number of amino acids will have what
• Most of the time, denatured proteins having a higher number of amino acids will have a bigger size and a higher Mr - molecular mass
(SDS-PAGE) stand for
Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis (SDS-PAGE)
what is used to seperate proteins based on size or mr
Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) as well as size exclusion chromatography can be used to separate proteins based on their size or Mr .
To measure the exact Mr of proteins whats used
to measure the exact Mr of proteins a mass spectrometer should be used.
