This document discusses quantitative mass spectrometry techniques for protein quantitation. It describes both labeling approaches, such as stable isotope labeling with amino acids in cell culture (SILAC) and isobaric tags for relative and absolute quantitation (iTRAQ), as well as label-free approaches including intensity-based measurements, spectral counting, and normalized spectral abundance factor. The labeling approaches involve metabolic, enzymatic, or chemical labeling of proteins prior to mass spectrometry analysis, while label-free methods quantify proteins based on spectral properties without labeling.
Introduction
Proteins
Function Of Protein And Their Properties
Protein Isolation And Purification
Methods Of Cell Lysis
Steps Of Protein Characterisation:
Determination Of Protein Concentration
Biuret Reaction
Lowry (Folin-Lowry) Method
UV- Spectroscopy
Assessment Of Protein Purity
SDS -Phage
Immunoblot
Surface Charge Analysis
Isoelectro Focusing
Ion Exchange Chromatography
Size, Shape And Conformation Analysis
2d-Electrophorasis
X-Ray Crytalliography
Protein Structure and Sequence Analysis
Edman Sequencing
Conclusion
References
Zymography is an electrophoretic technique for the detection of hydrolytic enzymes, based on the substrate repertoire of the enzyme. ... Zymography also refers to a collection of related, fermented products, considered as a body of work.
Analysis Analysis Analysis Analysisof the entire entire entire protein protein proteinproteincomplementcomplement complement complement of acell, cell, tissue, tissue, tissue, or organism organism organism under under aspecific, specific, specific, defined defined set of conditions conditions conditions .
• Relies Relies Relies on 3basic technological technological technological technological technological cornerstones cornerstones cornerstones cornerstones
• MethodMethod MethodMethod to fractionatefractionate fractionatefractionate fractionatefractionate complexcomplex complex protein/protein/ protein/ protein/ peptide peptide peptidemixturesmixtures mixtures
• MS to acquire acquire the data data necessary necessary to identify identify identifyidentifyindividual individual individual individualproteins proteins
• Bioinformatics Bioinformatics Bioinformatics Bioinformatics Bioinformatics Bioinformatics Bioinformaticsto analyze analyze and assemble assemble the MS data
Introduction
Proteins
Function Of Protein And Their Properties
Protein Isolation And Purification
Methods Of Cell Lysis
Steps Of Protein Characterisation:
Determination Of Protein Concentration
Biuret Reaction
Lowry (Folin-Lowry) Method
UV- Spectroscopy
Assessment Of Protein Purity
SDS -Phage
Immunoblot
Surface Charge Analysis
Isoelectro Focusing
Ion Exchange Chromatography
Size, Shape And Conformation Analysis
2d-Electrophorasis
X-Ray Crytalliography
Protein Structure and Sequence Analysis
Edman Sequencing
Conclusion
References
Zymography is an electrophoretic technique for the detection of hydrolytic enzymes, based on the substrate repertoire of the enzyme. ... Zymography also refers to a collection of related, fermented products, considered as a body of work.
Analysis Analysis Analysis Analysisof the entire entire entire protein protein proteinproteincomplementcomplement complement complement of acell, cell, tissue, tissue, tissue, or organism organism organism under under aspecific, specific, specific, defined defined set of conditions conditions conditions .
• Relies Relies Relies on 3basic technological technological technological technological technological cornerstones cornerstones cornerstones cornerstones
• MethodMethod MethodMethod to fractionatefractionate fractionatefractionate fractionatefractionate complexcomplex complex protein/protein/ protein/ protein/ peptide peptide peptidemixturesmixtures mixtures
• MS to acquire acquire the data data necessary necessary to identify identify identifyidentifyindividual individual individual individualproteins proteins
• Bioinformatics Bioinformatics Bioinformatics Bioinformatics Bioinformatics Bioinformatics Bioinformaticsto analyze analyze and assemble assemble the MS data
MASS SPECTROMETRY IN THE FIELD OF FOOD INDUSTRYErin Davis
This is a powerpoint presentation solely to give a brief idea about the role of Mass Spectrometry (MS) which is one of the powerful analytical technique.This presentation describes the role of Mass Spectrometry in the field of food industry.These slides deals with the basic principle,working,components,detailed analysis etc.
Harnessing The Proteome With Proteo Iq Quantitative Proteomics Softwarejatwood3
Learn how successful researchers are using ProteoIQ to streamline their proteomic data analysis.
Centralize data analysis on a single software platform
Most laboratories have multiple MS platforms with different software packages. ProteoIQ simplifies data analysis as a vendor independent software platform supporting qualitative and quantitative analysis.
Learn how to achieve robust peptide and protein quantification
ProteoIQ is the only commercial software platform supporting all popular forms of quantification. Learn how ProteoIQ performs protein and peptide quantification using isobaric tags, isotopic labels and label free methods including intensity based peptide profiling.
Elucidate biological significance
Learn how to integrate biological databases with ProteoIQ. Quickly move from MS results to the discovery of novel biological insights through an integrated biological annotation pipeline.
Accurate data are needed on threshold doses for allergenic foods. Data are also needed to demonstrate that available methods of analysis can detect and quantify allergens in foods at or around these threshold levels and that they are robust and fit for purpose. This is of major concern as no curative treatment is currently available for food allergy and accidental ingestion of the culprit food can lead to severe clinical symptoms. Elimination of the problem food ingredient from the diet reduces the risk of allergic reactions but this can lead to other issues such as deficiencies, eating disorders and growth retardation. Emergency medication is available including Antihistamines (H1 blockers), EpiPen (adrenaline-autoinjector) and Corticosteroids. This presentation describes investigations into peanut allergens and their quantitation in highly complex samples.
Carlos Afonso, Université de Rouen, Laboratoire COBRA, Plateau technique C2iorga
In this presentation, Carlos Afonso describes the analysis of polymers and petroleum by ion mobility mass spectrometry and utilises novel sample introduction techniques such as the Atmospheric Solids Analysis Probe (ASAP).
Join Brian Searle on an illustrated tour about interpreting MS/MS peptide spectra. On this tour you will first see how you can relate mass spectra to peptides. Next you see why the SEQUEST software was developed to interpret these spectra as peptides. Next you will see other software approaches have been developed and how combining approaches produces even better results.
Protein qualitative analysis based on mass spectrometry explores protein expression within organisms. Mass spectrometry offers highly efficient, robust, and accurate results and is one of the core technologies for proteomic research. Protein identification is a common topic for biochemistry research, and mass spectrometry is considered one of the most useful techniques that solve this issue. Two major strategies that are widely used for protein identification by mass spectrometry are MALDI-TOF-based protein fingerprinting and LC-MS/MS-based peptide sequencing. Meanwhile, LC-MS/MS reserved higher sensitivity and ability than MALDl-TOF and can accurately identify multiple protein components from a single sample. https://www.creative-proteomics.com/services/protein-identification.htm
Edman Degradation is one of the N-terminal amino acid sequence analysis methods for peptide chains/proteins sequencing. The protein is reacted with PTC under weakly basic conditions and then treated with an acid to free the amino-terminal residue of the peptide chain in the form of PTH-AA for subsequent analysis. Peptide Mapping analysis is an effective method for rapidly localizing protein sequences and is a commonly used strategy in protein identification. The method uses mass spectrometry for peptide analysis and compares the obtained spectra with a protein database to obtain amino acid information. De Novo Protein Sequencing is a method based on the enzymatically cleaved peptides that exhibit regular fragmentation in mass spectrometry to obtain amino acid information from the mass differences in regular mass spectral peaks. https://www.creative-proteomics.com/services/proteomics-service.htm
Edman Degradation is one of the N-terminal amino acid sequence analysis methods for peptide chains/proteins sequencing. The protein is reacted with PTC under weakly basic conditions and then treated with an acid to free the amino-terminal residue of the peptide chain in the form of PTH-AA for subsequent analysis. Peptide Mapping analysis is an effective method for rapidly localizing protein sequences and is a commonly used strategy in protein identification. The method uses mass spectrometry for peptide analysis and compares the obtained spectra with a protein database to obtain amino acid information. De Novo Protein Sequencing is a method based on the enzymatically cleaved peptides that exhibit regular fragmentation in mass spectrometry to obtain amino acid information from the mass differences in regular mass spectral peaks. https://www.creative-proteomics.com/services/proteomics-service.htm
Peptide Mass Fingerprinting (PMF) and Isotope Coded Affinity Tags (ICAT)Suresh Antre
Analytical technique for identifying unknown protein. The peptide mass are compared to database containing the theoretical peptide masses of all known protein sequences.
Mascot is a software package from Matrix Science that interprets mass spectral data into protein identities.
In this presentation we will study about MASCOT and also on how to use it.
Similar to proteomics, mass spectrometry, science, bioinformatics, electrophoresis, liquid-chromatography 3.ph d coursework-proteomicsclass-21dec2012 (20)
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
3. Quantitation Bias
Quantitative mass spectrometry in proteomics: a critical review. Analytical
and bioanalytical chemistry, 389(4), 1017–31. doi:10.1007/s00216-007-
8. Stable Isotope Labeling with
Amino Acids in Cell
Culture(SILAC)
http://www.biochem.mpg.de/mann/SILAC/index.html
9. Isobaric tags for relative and
absolute quantitation (iTRAQ)
Analysis of protein complexes using mass spectrometry
Nature Reviews Molecular Cell Biology 8, 645-654 (August 2007)
12. Overview
Less label, more free: approaches in label-free quantitative mass
spectrometry. Proteomics, 11(4), 535–53. doi:10.1002/pmic.201000553
13. Spectral Counting (SpC)
More abundant peptides will be
selected for fragmentation and will
produce a higher abundance of
MS/MS spectra, and is therefore
proportional to protein amount in data-
dependent acquisition
SpC = the number of MS/MS spectra
per peptide
14. Normalised Spectral Index(SIn)
SIN = a normalised spectral
index
To convert SI into SIn, SI is normalised for
variations between protein amounts across
data sets by dividing the SI for protein k by the
sum of the SI for all proteins in a replicate, and
is further normalised by dividing by the length
of a protein to account for the expectation that
15. RSC
RSC, = log2 of a ratio of abundance
between two samples
16. Protein Abundance Index
(PAI)
An estimate of the protein abundance
in a sample can be calculated using
the protein abundance index (PAI),
which is defined as the number of
observed peptides in the experiment
divided by the number of observable
tryptic peptides for each protein within
a given mass range of the mass
spectrometer employed
17. Exponentially modified
Protein Abundance Index
(emPAI)
which is directly proportional to the
protein content in a sample
The protein content can be calculated
in terms of a molar percentage by
dividing the emPAI value of a protein
by the sum of all emPAI values
multiplied by 100
18. Absolute Protein Expression
(APEX)
Absolute Protein Expression (APEX) is a
modified spectral counting technique that
takes into account the number of observed
peptide mass spectra for a protein and the
probability of the peptides being detected by
the MS instrument
The key feature of APEX is Oi, a correction
factor for the expectation of observing a
tryptic peptide in an experiment, which is
calculated by a machine learning
classification algorithm based on peptide
length and amino acid composition. This
technology is based on the findings of Mallick
and colleagues in a study involving the
prediction of proteotypic peptides
19. Normalised Spectral
Abundance Factor (NSAF)
Normalised Spectral Abundance Factor
(NSAF) provides an improved measure for
relative abundance by taking into account the
length of the protein, which is calculated by
dividing the SpC for a protein by its length (L)
This value is then normalised by dividing by
the sum of all SpC/L for all proteins in an
experiment
The dynamic range for NSAF values is
approximately four orders of magnitude, and
abundance changes as low as 1.4-fold can
be detected
