Functional Proteomics: Comparing Studies from 10 Years Ago to Today

This report provides a comprehensive comparison of proteomics studies, contrasting methodologies and technologies used in the field over the last ten years. It begins by defining proteomics and its significance, emphasizing the role of mass spectrometry (MS) as a core tool for protein analysis. The report traces the evolution of MS techniques, from early methods to advancements like MALDI and ESI, and the development of sophisticated mass analyzers. It discusses the challenges of analyzing complex biological samples and the need for protein fractionation techniques like SDS-PAGE and 2D-gel electrophoresis. The report further examines the emergence of techniques such as DIGE, MudPIT, and label-free quantitative methods, highlighting their applications in disease research, particularly in cancer and diabetes. The report also explores the use of nano LC-MS/MS and the bottom-up strategy. The study concludes by emphasizing the shift from gel-based to gel-free approaches and the increasing use of advanced technologies for rapid protein sequencing and data analysis. It provides a detailed overview of the evolution of proteomics research and its impact on the understanding of biological processes.

Running Head: FUNCTIONAL PROTEOMICS
FUNCTIONAL PROTEOMICS
Name of the Student
Name of the University
Author Note

1FUNCTIONAL PROTEOMICS
COMPARE AND CONTRAST THE WAYS IN WHICH PROTEOMICS STUDIES
ARE PERFORMED IN CURRENT LITERATURE AS OPPOSED TO THESE
PERFORMED TEN YEARS AGO.
ABBREVIATIONS: MS, Mass spectrometry; ESI, Electron spray ionization; MALDI.,
Matrix-assisted laser desorption/ionization; Q-Q-ToF, hybrid quadruple time-of-flight; ToF-
ToF, tandem time-of-flight; HPLC, high pressure liquid chromatography; MALDI-TOF,
Matrix associated laser ionization –time of flight; PMF, peptide mass fingerprinting; LC-
MS/MS, liquid chromatography coupled with tandem mass spectrometry; PMF, peptide mass
fingerprinting; DIGE, differential in gel electrophoresis; Mud PIT, multidimensional protein
identification technology; RP, reverse phase, iTRAQ, isbaric tag for relative and absolute
quantification ; DDA ,data dependent acquisition; MRM ,multiple reaction monitoring; SCX ,
strong action exchanger; SWATH ,Sequential window acquisition of all theoretical mass
spectra; ICAT, isotope- coded affinity tag;
KEYWORDS: shot gun proteomics/ Mass analyzer/ label free quantitative method/ Mass
spectrometry.
TOTAL NUMBER OF WORDS: 2009
ABSTRACT
Proteome are the modified forms of the collection of protein. Proteomics is the study
of proteins on a large scale. A proteome is also defined as a protein set that is being produced
in an organism. It is used for the identification of the patterns of the protein expression at a
particular time in trigger to a certain stimulus, along with this it also helps in the
determination of the protein networks that are functional that is exists at the stage of the
whole organism, tissue or cell. The comparative analysis of the protein which are at a normal
stage to the proteins that are present in the cancerous cell in order to determine the
biomarkers that are proteinaceous in nature and are linked with cancerous cells. The most
important tool for performing proteomics is Mass spectrometry and it is being used for a long
time. It helps in the determination of the polypeptide mass and their features or the type of the
post translational modifications. However, the shotgun proteomics and the detection of the

2FUNCTIONAL PROTEOMICS
protein based on MS are very confined to tiny molecules that are also thermostable until there
was evolution of the techniques like the matrix-assisted laser desorption or ionization
(MALDI) and electrospray ionization (ESI). This further stimulated the invention of the mass
analysers and a number of complicated instruments like, the hybrid quadruple time-of-flight
(Q-Q-ToF) and the tandem time-of-flight (ToF-ToF). Thus this review highlights on the way
proteomics studies have evolved along with the advancements of the technology in the field
of proteomics in the last 10 years.
SIGNIFICANCE OF THE PAPER:
This report analyses the advancements and development in the field of proteomics
from the last 10 years. It shows a huge range of technologies that are associated with
proteomic and which has been used because of their higher resolution and accuracy. It gives
information about the recent ways in which the research of proteomics is conducted, the
obstacles and the profits benefits of the study in clinical proteomics.
INTRODUCTION
Previously, the formation of polypeptide was done manually and the complete
analysis of the protein was very critical and troublesome. The analysis of the protein needs
the dissociation of maximum number of peptides from a number of digested part of the target
protein along with the help of the proteases that has certain specific properties that have the
ability to gather the matched fragments that cover up the complete sequence (Sarbu, Ghiulai
and Zamfir 2014). The protein sequencing is done using the Edman Degradation. There are
certain specialist advances such as the use of the novel valves to manage the harsh chemicals,
the beginning of the HPLC or high pressure liquid chromatography, and the use of
immobilization of the sample which were all added in the first gas phase that helps to
increase the susceptibility and also allowed the automatic collection of data and their analysis
(Sun, Chen and Yao 2013). On the other hand, the instruments that are used in the present

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times have an affectability of the molecules that are as low as PicoMole and shows faster
advancements in the innovations related to DNA. For instance, the enhancement of the
cloning and the dideoxynucleotide sequencing, and restriction mapping has helped the
development of protein science which was not possible in the past (Leitne 2017).
Mass Spectrometry was earlier used for the detection and the quantification of the
separated protein. In the recent times the technology has faced a number of technical
advancements in the terms of resolution and accuracy that gives a chance for the discovery of
a number of species that are less abundantly available (Leitne 2017). However, the large
number of more abundant species makes it difficult for the detection of the biomarkers. It is
also very difficult to identify the protein in their complete abundance from a sample mixture.
The analysis of the protein in a serum sample or any biological sample obtained from the
patients is very difficult because of their dynamic range (Sun, Chen and Yao 2013). Hence
the characterization of these samples is done on the basis of the low abundance of the more
abundant molecule. However, the species that are highly abundant might suppress the
detection of the low abundant molecules. As the availability of the biomarkers in the sample
is low thus, in order to lower the complexity of the specific sample it is necessary to remove
the protein molecules that are highly abundant (Savaryn et al. 2013). Along with this, the
ability to analyse the protein molecules that are less abundant the protein fractionation is a
mandatory process before the complete identification of the protein. The removal of a protein
molecule like albumin which is highly abundant as well as heavy is done by the use of the
fractionation techniques. A few biomarkers are linked to the abundant protein molecule
which can be a risk at the time of removal, but every protein needs the fractionation step that
are done using a number of different approaches (Li,Wang and Chen 2017).
The most used method for the fractionation of protein is based on the SDS-PAGE.
This includes the separation of the protein on the basis of the molecular weight under the

4FUNCTIONAL PROTEOMICS
denaturing conditions. This is used for a long time and has a standardized protocol for the
digestion of the protein. One of the major drawback of this process is that the extraction of
the peptides from the gel is a difficult process (Sarbu, Ghiulai and Zamfir 2014). As a result a
number of other processes has been designed to extract the protein from the gel. It is very
evident that the optimized protocol needs a number of steps that involves incubating the
fragments with trifluoroacetic acid at varying concentration. Finally in the last step there is
mixing of the peptides followed by MALDI-TOF or the LC-MS/MS (Yao, Sun and Deng
2018).
At present times there has been a substitute for the one dimensional gel
electrophoresis that is the serum proteomics for the analysis of the biomarkers. In this case,
the separation of the protein is done by an electronic apparatus and the fractionation is based
on the molecular weight of the protein. After the proteins are run in gel the fractions are
collected (Donnarumma et al. 2016). After the removal of SDS from these fractions they are
then analysed by the MALDI or LC-MS/MS. The important part of the method is that it helps
in the separation of the protein molecules that are bound to the carrier protein molecules
(Marx 2013). Apart from the 1D-gel electrophoresis there is development of the 2D-gel
electrophoresis that gives a better resolution. A large of spots are obtained in 2D-gel
electrophoresis and from that the desired protein can be identified. Another benefit of the
process is that a large number of the protein isoforms can be identified. The identification of
the spots is done using comassie blue and silver stain as well the use of the antibodies. This
method is helpful for studying the immunoproteomics which is the study of autoimmune
disorders, also in identifying the natural antibodies that are produced for the tumor by the
immune system. However, the study of immunoproteomics is very specific and is selective of
the antibodies (Monaci et al. 2018).

5FUNCTIONAL PROTEOMICS
Another method that is used as a substitute for the proteomics study is DIGE or
differential in gel electrophoresis in which the proteins are labelled with the fluorescent dyes
and are mixed together before they are subjected by the 2D-gel electrophoresis (Marx 2013).
This procedure allows the samples for the comparison with other proteins under exact
experimental situations and the specific spots can be located and separated on the basis of the
emission spectra. The specific spots are detected by MS analyses by the peptide mass
fingerprinting (PMF) or by the use of the tandem mass spectrometry (MS/MS).The spots that
has to be analysed must undergo enzymatic digestion before the MS analysis (Ma et al.
2013).
In the last 10 years, there has been an increase in the use of the functional proteomics
in the treatment of various diseases like cancer or diabetes. In order to reduce the burden of
diabetes a number of conventional proteomics method are used, that involves gel-based
method, that exhibits low levels sensitivity and are much more robust and are typically used
in the discovery of biomarker. The other benefit is in the use of cancer patients that help in
the identification of the deposition of the tumor cells that can help in the determination of
cancer (Altelaar, Munoz and Heck 2013). The main benefit of the method is the analysis of a
large number of proteins at one time. The method is also very economical in the field of
clinical studies because the analysis can take place at a large scale by the taking small amount
of the samples. However, this method cannot be beneficial at the discovery stage of the
analysis of the protein as the post translational modifications of the proteins were not
considered earlier.
An effective method for the separation and the fragmentation of the peptides is Mud
PIT or multidimensional protein identification technology (Dittrich et al. 2015). This method
is an amalgation of the strong action exchange chromatography and the reverse phase. Then
the eluted fraction is subjected to the Reverse Phase Chromatography and at the end of the

6FUNCTIONAL PROTEOMICS
elution they are analysed by MS/MS. There are a number of tagging methods that are used
for the quantification of the proteins (Monaci et al. 2018). The label free quantitative
methods, is a dependent on the features from LC-MS/MS. This includes the peak area that is
required for the high consistency of the chromatographic separation or SWATH. This tool is
very robust that can be used for the quantification as well as the identification of the proteins
that are matrisomal in nature. The validation of the protein is done by the use of software that
uses the statistical analysis along with different combination in the LC MS/MS analysis
(Barallobre et al. 2013). The quantification of proteomics includes the annotation of the
peptide, the selection of the target protein for quantification using the help of an unique
identifier for instance, Uniport, Ensembl . The collection of the databases which is obtained
allows the exploration of the potential isoforms of the protein and the post translational
modification of the specific protein. It also helps in understanding the sequence and the
probable destination of the sequence of amino acid variant that is caused because of a single
nucleotide polymorphism (Kang, Lee and Lee 2016).
CONCLUSION
The recent studies have considered the approach which is gel-based to the gel-free
mono-dimensional chromatography. There is more use of the nano LC-MS/MS for the
resolved and the protein data that is fast sequenced. These approaches require protein
fractionation and also constitute a complex strategy that is called as the bottom up strategy.
Intact protein can be analysed by the use of the top down using the MALDI or he LC-MS/MS.
This method has been coupled to the 3D –separation of the proteins along with the MS
analysis of the complete protein. MRM or multiple reaction monitoring, which is also called
as the selective reaction monitoring where the target ions are identified for the fragmentation.

7FUNCTIONAL PROTEOMICS
Thus, it can be concluded that the recent developments in the proteomics is very critical and
has helped for the entire characterization of the protein.

8FUNCTIONAL PROTEOMICS
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