good about protein production

1. ICAR- Central Institute of Fisheries Education
ISO 9001:2008 Certified
(University under Sec.3 of UGC Act, 1956)
Panch Marg, Off Yari Road, Versova, Andheri (W)
Mumbai – 400061
APPLICATION OF PROTEOMICS
IN ENVIRONMENTAL STUDIES
FBT – 608
ADVANCES IN PROTEOMICS AND
METABOLOMICS
AMRITA MOHANTY
FBT-PB1-01

2.  The term ‘proteome’ was first coined in 1994 by an Australian post doctoral fellow named Mare wilkins.
 Proteome refers to the total set of proteins expressed in a given cell at a given time.
Genomics DNA (gene)
Transcription
Transcriptomics RNA
Translation
Proteomics PROTEIN
Enzymatic reaction
Metabolomics METABOLITE
What is proteomics ?
To study the dynamic
protein products of the
genome and their interaction
A large scale characterization
and functional analysis of the
proteins expressed by a
genome

3. PROTEOMICS
 Proteomics is the study of the proteome; it uses various technologies ranging from genetic analysis to mass spectrometry.
 It also assesses activities, modifications, localization and interactions of proteins in complexes.
 With the development of proteomics techniques, proteome analysis provides a fast, non-invasive diagnostic tool for patients
with various diseases.
 The advent of highly sensitive proteomic technologies can identify proteins associated with development of diseases well before
any clinically identifiable alterations.
 MS has a high resolving power and identifies proteins with more accuracy.

4. PROTEOMICS OBJECTIVES
 Protein/peptide separation
 Identification and characterization of resolved proteins
 Data analysis and applications

5. TYPES OF PROTEOMICS
 Structural proteomics –
The ultimate aim of this proteomics is to build a body of structural information that will help predict the probable structure and potential
function for almost any protein from knowledge of its coding sequence.
 Functional proteomics –
It refers to the use of proteomics techniques to analyse the characteristics of molecular protein – networks involved in a living cell.
 Expression proteomics –
It refers to the quantitative study of protein expression between sample differing by some variable.

6. WORKFLOW OF PROTEOMICS
Rebeca et al., 2016

7. PROCEDURE OF PROTEOMICS
 Separation of proteins
one dimensional electrophoresis
2-D electrophoresis (modern)
Multidimensional HPLC (modern)
 Analysis of proteins
Mass spectrometry (modern)
 Database utilisation

8. PROTEOMICS TECHNIQUES
 Gel based
i) SDS-PAGE
ii) 2-DE
 Off gel base
i) LC (SCX, RP-LC, Immuno affinity)
 Quantitative proteomics
i) iTRAQ, ICAT, SILAC
 MS (Mass spectrometry)
i) MALDI, LC-MS, CE-MS

9. APPLICATIONS OF PROTEOMICS
 Protein sample identification/confirmation
 Detection of post-translational modifications
 Detection of ammino-acid substitution
 Mass fingerprint identification of proteins
 Nutrition research
 To identify unknown protein of interest
 Protein biomarker
 Study of tumor metastasis
 Proteome profiling in environmental microbiology and
biotechnology.
Sinha et al., 2020

10. PROTEOMICS IN ENVIRONMENT PROSPECTS
 With the development of the global industry and growth of human population, thousands of manmade chemicals are released to
environment by agriculture, transport, industries and other activities.
 Proteomics approaches have been applied in environmental science as they are capable of detecting subtle changes in the level
and structure of individual proteins in response to the environment stresses.
 The proteomics approaches have been applied in environmental research from microorganism, plants to invertebrates and
vertebrates.

11. Continued……
 Proteomics provides an insight into various mechanism of toxicity and can be further divided into two areas –
a) Mechanism study of proteomic response to stresses, which can help us understand how the cells respond to
environment stresses and some organisms could resist extreme or toxic environmental stresses.
b) Screening environmental samples with proteomics approaches, which can monitor concerned pollutants.

13. STUDIES ON PROKARYOTIC MICROORGANISM
 For prokaryotes, Escherichia coli (E. coli) and Bacillus subtilis have been used as the gram negative and positive model strain
separately to study the normal environmental stress, such as pH, oxidation, etc. and they found 22 proteins were found to be
regulated by pH, these proteins included periplasmic proteins, membrane proteins, and three other proteins whose functions
were not clear.
 Further, when exposed to external acid (pH was from 4.9 to 6.0), several acetate induced proteins were increased (e.g., LuxS
and YfiD). But, (RibB) the homolog protein of LuxS, was not induced by acetate although it was also induced at low pH,
showing a different pH induced mechanism (Chu et al., 2009).
 When exposed to alkaline environment, tryptophanase (TnaA) became one of the most highly expressed proteins in the cells,
which is a versatile enzyme, deaminates serine and cysteine. Besides TnaA, three more additional enzymes were reported to be
induced under high pH environment (namely AstD, GabT, and CysK) with different functions (Stanick et al., 2005).
 In E. coli, two aconitases AcnA and AcnB were reported to be induced by oxidative stress. The mutant E. coli strains which
lesion the gene acnA or acnB or both were cultured, and their proteome were compared with the wild strain after exposure to
peroxide. At least 11 polypeptides were changed in the acnB mutant, and 4 of them have been identified by sequence analysis,
i.e., ODH, YedA, dihydrofolate reductase, and SodA (Cunnigham et al., 2002).

14. Continued ……
 Delftia acidovorans MC1 is able to grow on chlorophenoxy herbicides such as 2,4-dichlorophenoxypropionic acid (2,4-
DCPP) and 2,4- dichlorophenoxyacetic acid as sole sources of carbon and energy, and the responses of Delftia acidovorans
MC1 to high concentration of chlorophenoxy herbicides was studied at the protein level (Benndorf et al., 2008).
 In the identified proteins, many were involved in the metabolism of 2,4-DCPP, such as chlorocatechol 1,2-dioxygenases
(TfdC and TfdCII), 2,4-DCP hydroxylase (TfdB), and so on.
 Under the environmental stresses, some proteins were either up regulated or down regulated, and the changes in expression
level could be studied to discover the function of the proteins.
 2D-PAGE as a high throughput analysis technique was applied to study the global proteome of E.coli after benzoic
acid treatment the largest protein expression changes were discovered to be the protein ompF (including 3
proteins) and protein znuA (Yan et al., 2002).

15. STUDIES ON EUKAROTIC ORGANISM
 Fission yeast Schizosaccharomyces pombe and budding yeast S. cerevisiae have been used as the eukaryotic cell models to
study the cellular reactions to the toxic environmental stress because of their easy to handle genetic manipulation and their
complete genome information.
 The proteomic response of S. cerevisiae to oxidative stress was investigated and 0.4 mmol/L of H2O2 was used to treat the
cells and the proteomic study showed that at least 115 proteins were stimulated, whereas 52 proteins were repressed by this
treatment. Out of these 115 proteins only 71 protein, further they were sorted into seven different functional classes: antioxidant
defense, heat shock proteins, proteases and proteasome subunits, translation apparatus components, carbohydrate metabolism
enzymes and enzymes involved in amino acid metabolism (Godon et al., 2000).
 S. cerevisiae was exposed to 0.9 mmol/L of sorbic acid at pH 4.5, and the changes in protein presented that 10 proteins were
up regulated and 3 proteins were down regulated. Among those up regulated proteins, most were stress proteins and chaperones
(Nobel et al., 2001).
 They reported proteomic responses of S. cerevisiae to cadmium stress were analyzed after S. cerevisiae were cultured with 0
μmol/L, 25 μmol/L, 50 μmol/L, 75 μmol/L, 100 μmol/L, and 200 μmol/L of cadmium sulfate for 2–5 days, 54 induced and 43
repressed proteins were identified. Hence, they revealed that eight enzymes of the sulfur amino acid and GSH biosynthesis
pathway were strongly induced, and several proteins with antioxidant properties were also induced (Vido et al., 2001).

16. Continued…..
 The cellular responses of the S. pombe to cadmium were also studied using an integrated proteomic strategy. Amino acid
coded mass tagging (AACT) was integrated with LC-MS/MS to study the cellular response to cadmium. A total of 1133
proteins were identified, and among the 319 quantitated proteins 106 were up regulated and 55 were down regulated. Most
prevalent class of the up-regulated proteins included heat shock proteins, oxygen and radical detoxification, and stress response
proteins (Bae et al., 2004).
 Teixeira et al., 2005 reported that S. cerevisiae was incubated with 0.3 mmol/L of 2,4-D and a total of 26 protein spots whose
expression increased more than 2 times under the 2,4-D stress were identified, corresponding to 22 different proteins. The
decreased protein under 2,4-D stress was identified as a single protein Ado1p and three proteins that increased during exposure
to 2,4-D were involved in cell protection against environmental stress –
(i) an antioxidant enzyme Ahp1p, with a specific role in the reduction of alkyl hydroperoxides.
(ii) Ssb2p, belonging to the heat shock proteins of the Hsp70 family, whose function is promoting proper protein
folding.
(iii) Hsp12p, a small heat shock molecular chaperone over expressed under a wide range of environmental stresses.

17. STUDIES ON INVERTEBRATES
 Mussels have been widely used as the indicator of marine pollution due to its capacity of bioaccumulating and concentrating
organic and metallic pollutants.
 The proteomic profiles of M. edulis when exposed to several marine pollutants; diallyl phthalate (DAP), 2,2’,4,4’-
tetrabromodiphenl ether (PBDE-47), and bisphenol-A (BPA) were identified and found 170 spots showed a significant
increase or decrease in protein abundance in the 2-D electrophoresis maps from the groups exposed to pollutants (Apraiz et al.,
2006).
 In addition, the individual proteomic profiles to specific stresses as PESs (Protein expression signature) could be used as the
signature of the specific stress to monitor the environment.
 In this study, the PESs of M. edulis, which is composed of 13 proteins including oxidation, amino acid metabolism,
detoxification, protein degradation, organelle biogenesis, and protein folding were used to distinguish the clean sample from that
of the polluted marine water samples (Amelina et al., 2007).
 Thus, the PESs of M. edulis have been used to monitor polluted marine water.
 Some proteins such as metallothionein, cytochrome p450, in “sentinels” marine species act as excellent indicators for stress in
environment and potential health threats for humans (Stewart et al., 2008).

18. Continued……
 Microplastic is also a critical issue in the aquatic system and after they undergo chronic exposure these plastic particles get
bioaccumulated basically, in invertebrates such as crustaceans, barnacle, polychaete worms, mussel and amphipods (Graham
and Thompson, 2009).
 Advances in techniques in proteomics might hold promise for upcoming reviews in this area by techniques such as pyrolysis
combined with chromatography, mass spectrometry and scanning electron microscopy with energy dispersive X-ray
spectroscopy (Boumester et al., 2015).
Lopez et al.,
2019

19. STUDIES ON VERTEBRATES
 Because the liver and kidney are both common sites for toxicity within the body, several proteomic studies have been
performed to define changes in the liver cells or kidney cells in response to damage.
 Anderson and colleagues (2004) have described the xenobiotic effects in rodent liver by using 2D gel electrophoresis
technologies, a prototype Molecular effects Databases and this database can detect, classify, and characterize a broad range of
liver toxicity mechanisms.
 Human amnion epithelial cells (FL cells) were used to study the cellular responses to Benzo[α] pyrene (B[a]P), which is a
prototype of polycyclic hydrocarbons (PAHs) and a potent procarcinogen generated from the combustion of fossil fuel and
cigarette smoke (Gao et al., 2004).
 In this study, the proteomics analysis of human intestinal Caco-2 cells treated with the wastewater effluent (nonylphenol and
lipopolysaccharide), they induce overexpression of specific proteins, namely elongation factor 1 β and enolase 1 were found,
which suggested that specific proteins can be used as biomarkers for the risk assessment of water and wastewater (Han et al.,
2007).

20. CONCLUSION
 The development of proteomics relies on the development of a series of technologies, from protein separation and identification
to gel image capturing and comparing technologies, and any of them could be the bottle-neck of the development of proteomics.
 The main challenges of proteomics development are high sample throughput and reproducibility, which are also the limitation
of the application of proteomic technology in environmental researches.
 Despite such challenges, the application of proteomic approaches in environmental science developed rapidly in recent years
due to its advantages of a global approach for understanding the complex mechanisms of environmental stresses and sensitive
capability of monitoring an individual or a group of pollutants.
 With the development of high reproducibility and throughput technologies, wide applications, and increased demand in
environmental studies, the cost is expected to be reduced in the future, which would in return promote proteomics applications
in environmental sciences.

21. REFERENCES
 Chu, X., Hu, J. and Ong, S.L., 2009. Application of proteomics in environmental science. Frontiers of Environmental Science &
Engineering in China, 3(4): 393-403.
 Blankenhorn D, Phillips J, Slonczewski J L. Acid and base inducedproteins during aerobic and anaerobic growth of Escherichia coli
revealed by two dimensional gel electrophoresis. J Bacteriol, 2008, 181(7): 2209–2216.
 Amelina H, Apraiz I, Sun W, Cristobal S. Proteomics-based method for the assessment of marine pollution using liquid
chromatography coupled with two-dimensional electrophoresis. J Proteome Res, 2007, 6: 2094–2104.
 Han J, Takenaka M, Talorete T P, Funamizu N, Isoda H. Toxicity assessment of wastewater by proteomics analysis. Environ
Sci, 2007, 14(Suppl): 35–41.
 Apraiz I, Mis J, Cristobal S. Identification of proteomic signatures of exposure to marine pollutants in mussels (Mytilus
edulis). Mol Cell Proteomics, 2006, 5(7): 1274–1285.
 Stancik L M, Stancik D M, Schmidt B, Barnhart D M, Yoncheva YN, Slonczewski J L. pH dependent expression of periplasmic
proteins and amino acid catabolim in Escherichia coli. J Bacteriol, 2005, 184: 4246–4258.
 Bae W, Chen X. Proteomic study for the cellular responses to Cd2+ in Schizosaccharomyces pombe through amino acid coded
mass tagging and liquid chromatography tandem mass spectrometry. Mol Cell Proteomics, 2004, 3(6): 596–607.
 Anderson N L, Taylor J, Hofmann J P, Esquer-Blasco R, Swift S, Anderson N G. Simultaneous measurement of hundreds of liver
proteins: Application in assessment of liver function. Toxicol Pathol, 2004, 24(1): 72–76.
 Gao Z, Jin J, Yang J, Yu Y. Zinc finger proteins and other transcription regulators as response proteins in benzo[a]pyrene
exposed cells. Mutat Res, 2004, 550: 11–24.
 Yan J X, Devenish A T, Wait R, Stone T, Lewis S, Fowler S. Fluorescence two dimensional difference gel electrophoresis and mass
spectrometry based proteomic analysis of Escherichia coli.Proteomics, 2002, 2: 1682–1698.