1. Proteomic Techniques
All the various types of proteomics and biomarkers described in the previous section come down to the method/technology used to extract and identify proteomes, cells, or other substances. These techniques, also, caused the limitations seen most often. So, finding an ideal proteomic analysis procedure with a low margin of error is of prime significance.
1.1. Two-Dimensional Gel Electrophoresis (2DE)
Proteomic analyses were, first, carried out by the traditional two-dimensional gel electrophoresis (2DE), which separates sequentially, mid-range molecular weight proteins based on two distinct characteristics of the protein: size and charge
[1][43]. It has been mentioned that proteome analysis with 2DE techniques was later expanded with liquid chromatography-mass spectrometry (LC-MS) techniques. Despite the rapid advancement of MS-based proteomics, 2DE continues to play an important role in the areas of protein identification from organisms with no or incomplete genome sequences, alternative detection methods for specific proteomics modification, and identification of protein isoforms and modified proteins. This proves that there is a significant market for 2D gel-based proteomics, which is supplemented by traditional LC-MS techniques
[1][43]. The protein contents of tissue or biofluids are separated using two-dimensional gels and must be visualised using staining. Silver staining used to be employed for this purpose, but, as of late, other dyes such as Deep Purple and Sypro Ruby, or newer staining techniques, namely fluorescent labelling (CyDye), are being utilised
[2][91]. For protein labelling, the CyDye method employs three different fluorescent dyes with different emission spectra: Cy2, Cy3, and Cy5. Three differentially labelled samples are run on a single gel, and a gel image is generated for each of the three dyes, due to their distinct emission spectra. These images are, then, superimposed on top of one another, and differences in protein levels are analysed using sophisticated software such as Prognosis (Nonlinear Dynamics, Newcastle, UK) or DeCyder (GE Health, Little Chalfont, UK)
[2][91]. One significant advantage of this method is that the three differentially labelled samples produce perfectly matching gel images, removing gel-to-gel variability. The introduction of CyDye vastly improved reproducibility and detection sensitivity in the picogram range, due to the ability to run disease and non-disease samples, as well as internal control, on a single gel
[2][91]. The proteins can, later, be identified using MS, after differences in protein levels of candidate markers between disease and control groups have been determined
[2][91]. The best ‘snapshot’ of the protein repertoire of the cell or body fluid has come from 2DE separation. However, this technology, on its own, is limited because it has low throughput, is time-consuming, is labour intensive, and has difficulty detecting proteins with a basic charge or those smaller than 10 kDa
[3][92].
1.2. MS-Based Techniques
In proteomics, MS is the most-used protein identification technique. It is utilised for fingerprinting proteins and peptides, after which direct sequencing can be done
[4][5][93,94]. It helps in precisely determining the mass and charge (
m/z) of proteins and, as a result, identifying the exact progenitor proteins. MS instruments have substantially improved in recent years and are now widely used to detect even small samples, as they are sensitive to the picomole- femtomole range, necessary for oligonucleotide, glycoprotein, and other minuscule molecule detection
[1][43].
An ion source, a mass analyser, and a detector make up a mass spectrometer. Ionization sources, such as matrix-assisted laser desorption and ionization (MALDI) and ESI; mass analysers, such as time-of-flight (TOF), quadrupole, and ion traps; and fragmentation methods, such as collision-induced dissociation (CID), electron-transfer dissociation (ETD), and electron-capture dissociation (ECD), can all be used in various combinations in MS
[1][43]. The most widely used separation method for studying biological samples by MS or MS/MS (tandem mass chromatography) is high-performance liquid chromatography (HPLC)
[1][43].
The shotgun-MS method entails the separation of peptides by protein digestion in complex mixtures, tandem mass spectrometric analysis, and, finally, running the process through databases to identify the peptides. The initial step of MS is to lyse the sample and extract the proteins from the sample. This is followed by the digestion of the proteins into peptides, resulting in the yield of thousands of peptides, which are then enriched in various ways (for example, affinity resins or specific antibodies) or pre-fractionated, based on their physicochemical properties (such as charge or isoelectric point). These samples are, later, individually examined using reversed-phase liquid chromatography added to MS (LC-MS). Next, MS/MS or MS is connected to software, which analyses the identified ions based on their relative abundance and
m/z. Finally, these ions are uploaded/cross-verified in the databases, enabling the identification of the fitting peptides sequences
[1][43].
The dynamic range, rather than sensitivity, is the most difficult challenge in most MS approaches. To increase the amount of information that can be obtained from specific samples, the common proteins or peptides can be removed. Intriguingly, common proteins such as albumin can also act as carrier proteins, capturing a subset of proteins and peptides helping achieve optimal results
[4][93]. Recently, the use of MALDI and SELDI MS techniques have skyrocketed, due to their higher rates of accuracy.
1.2.1. MALDI-TOF
MALDI with TOF is a technique that uses laser excitation to present proteins in a matrix. In the MALDI-TOF detection, samples of interesting protein are immobilised on an energy-absorbing chemical matrix on a chip or plate. This method is followed by the analysis of the entire proteome, and presentation of the proteins coupled in a matrix to laser excitation. The peptides, which are ionised by this process, are, later, sent to the detector through a vacuum tube. Like the traditional MS, peptides are detected in order of their
m/z. Based on the time each peptide reaches the detector, a peptide fingerprint is formed that reflects the sample’s protein composition and the relative abundance of specific proteins. In many circumstances, guided proteolysis is employed to reduce the size of the peptide, to put the charge to mass ratio into the ideal range for the MS system in question. Peptide mass fingerprints are compared to huge, published databases and masses predicted by protein sequences to identify proteins or peptides
[4][5][93,94].
Another MALDI technique is MALDI-MS imaging (MALDI-MSI). Here, mass spectrometry imaging (MSI) combines immunohistochemistry, fluorescence microscopy, and MALDI-MS instrumentation, to provide a specific molecular image of numerous expressed proteins within a tissue sample. To perform MALDI-MSI, sections of biological tissues are introduced into a MALDI-MS instrument, and the ultraviolet-pulsed laser of the MALDI source is used to raster over a selected area, while acquiring mass spectra of the ablated ions at each image point
[4][5][93,94]. However, MALDI-MSI has not yet been used to map the transcriptome, including miRNA and other RNA-related molecules
[1][43].
There is now a broad field of clinical research to investigate this, known as tag-based mass spectrometric imaging (Tag-MSI) or targeted mass spectrometric imaging (Tag-Mass MSI), which supplements the MALDI-MSI
[1][43].
1.2.2. SELDI-TOF
SELDI is a refinement of MALDI that uses a selective surface to bind a subset of these proteins based on absorption, partition, electrostatic interaction, and/or affinity chromatography. Artificial intelligence techniques are needed to sort or mine the data and extract critical information because a SELDI proteomic profile can now have up to a million data points (high-resolution spectra of 400 ppm)
[5][6][94,95].
SELDI-TOF technique employs stainless-steel- or aluminium-based solid supports, with predetermined bait sections comprising a variety of surface-binding chemistries, such as hydrophobic, normal-phase, metal-affinity, cationic, or anionic, as bait in a 1–2 mm diameter area. It does not require prior purification or protein fractionation, and cell lysates or bodily fluids as tiny as 0.5 μL can be applied to these surfaces
[3][92]. Proteins and peptides are, selectively, kept on the protein chip after a wash phase; based on the bait used and the unique chemistry of each protein, they are then evaluated using mass spectrometry TOF technology. Similar to MALDI, ionized proteins and peptides are recorded when they impact the detection plate, and the variation in the journey time down the vacuum tube is recorded
[3][92]. It can profile proteins, regardless of their intrinsic hydrophobicity (a limitation of 2DE analysis), and is extremely sensitive in detecting proteins in the lower-molecular-weight range. Finally, data-mining methods examine low molecular weight (0–20 kDa) proteomic data streams generated by SELDI-TOF, sorting the data into homogenous (control or illness) groups
[3][92].
1.3. Protein Microarrays
Protein microarrays investigate how proteins are expressed and activated, thereby providing a functional view of protein networks
[5][94]. Microarray technology has allowed researchers to assess the expression of tens of thousands of genes in a single tissue sample and compare normal and abnormal gene expression, such as the difference in the regulation, during cancer formation, in malignant cells using antigen-antibody interactions.
Protein microarrays can be divided into two major categories: forward phase arrays (FPAs) based on cell lysate probing and reverse phase arrays (RPAs) based on antibody probing
[1][43].
In both forms of microarrays, a protein substrate is immobilised and queried using a tagged probe. Protein expression is, then, quantified based on the strength of the resultant signal. However, in RPAs, antibodies are immobilised, as bait molecules, to catch proteins from the biological milieu. FPAs, on the other hand, necessitate the immobilisation of cellular lysates, which are, subsequently, probed with particular antibodies for the proteins of interest
[5][94].
RPAs, unlike FPAs, do not necessitate the labelling of cellular protein lysates and, thus, provide a useful platform for biomarker screening, therapeutic monitoring, and pathophysiologic studies. Furthermore, the RPA is unique in its ability to analyse signalling pathways, utilising small numbers of cultured cells or cells isolated via laser capture microdissection (LCM) from human tissues obtained during clinical trials
[1][43].
The RPA platform has helped in identifying therapeutic targets, the study of disease progression, and profile signalling pathways, as well as suggesting prognostic indicators in OC. A major drawback of RPAs is that they necessitate the use of particular antibodies, so there remains a perpetual risk of non-optimal antigen-antibody interaction, which could lead to incorrect results. Therefore, these approaches should be considered purely exploratory
[1][5][43,94].
1.4. Mitochondrial Proteomics Methods
In eukaryotic cells, the mitochondrion is a highly specialised organelle with vital biological activities. It is a multifunctional organelle that is responsible for energy metabolism, oxidative stress, cell apoptosis, cell cycles, mitophagy, and communication with other organelles. Over the last few decades, research has led to the identification of mitochondria-related signalling pathways for insights into the mitochondrial mechanisms in tumourigenesis, identifying biomarkers for diagnosis and prognostic assessment, and discovering therapeutic targets for effective therapy
[7][96].
Since the first human placental mitochondrial proteome was analysed with MS in 1998, great advances in mitochondrial proteomics have been made
[8][97]. The reason for the probing into the mitochondrion is not only due to its multifunctional characteristics, but also because it is the only organelle other than the nucleus to contain independent DNA: mitochondrial DNA (mtDNA). There is compelling evidence that mtDNA influences tumour progression cell functions and susceptibility in the tumour microenvironment, and several studies have found that mitochondrial dysfunction is closely linked to OC
[9][98].
A study by Hecker et al. of mitochondria in cells with OC under an electron microscope showed that the loosened mitochondria have a moth-eaten appearance, which indicated their involvement in OC
[10][99]. Another study by Dier et al. reiterated this fact, by proving that altered mitochondrial fission dynamics in the mitochondria affected the phenotype of specific epithelial OC cells
[11][100]. Some mitochondrial proteins, such as Bcl2, p53, and galectin3, have also been reported to be therapeutic targets used to suppress mitochondria-related pathways in OC
[12][13][14][101,102,103]. As a result, quantifying mitochondrial proteome changes will shed light on any mitochondria-mediated pathophysiological changes in OC.
The importance of high-purity mitochondrial samples in proteomic analysis cannot be overstated. Many methods can be used for this purpose, including kit-based methods, free-flow electrophoresis, kit-based methods, and density-gradient centrifugation. Multidimensional protein identification technology (MudPIT) combines strong cation exchange (SCX) prefractionation, reversed-phase high-performance LC separation, and MS to increase the number of identified peptides and the power of peptide separation
[15][104]. After which, to isolate the mitochondria, quantify mitochondrial proteins, and quantify mitochondrial phosphoproteins, several quantitative proteomics methods can be utilised, such as the SILAC, ICAT, iTRAQ, tandem mass tags (TMT), and label-free methods.
Using these techniques, the mitochondrial proteome in OC has a quantitative reference map established, especially, for those that are platinum-sensitive (A2780) and platinum-resistant (A2780-CP20)
[16][105]. In addition, around 5115 mitochondrial expressed proteins (mtEP) have been identified
[17][106]. Further analysis of the mtEPs has revealed several potential biomarkers and signalling pathways in OC, such as CPT2, PKM2 (overexpressed in cancerous tissues), and HMGCS2.
The protein phosphorylation profile of mitochondria has also been identified, to understand the molecular mechanisms in pathophysiological conditions, thanks to significant advances in MS-based proteomics
[18][107]. For example, phosphorylated cofilin 1 (p-CFL1) was found to be highly expressed in paclitaxel-resistant OC cells and chemoresistant cases relative to chemosensitive ones from primary human OC tissue. As a result, it can be stated that mitochondrial proteomics in conjunction with clinical data are a valuable resource to develop a prognostic model in OC.
2. New Approaches in Proteomics
2.1. Targeted Proteomics
Targeted proteomics is a key technique that enables the validation and verification of biomarkers that have been discovered. It works with untargeted proteomics to complete the cycle of biomarker discovery and validation. It consists of two types, –multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM), which can facilitate quantitation accuracy during the validation and verification process of biomarker discovery
[19][108].
MRM is a traditional targeted quantitative proteomics approach that employs either triple quadrupole (QqQ) or quadrupole linear ion trap (QTRAP) instruments
[20][109]. The targeted distinctive precursor ions for peptides of interest are selected in the first quadrupole (Q1) and, then, transmitted into the collision cell (Q2) for fragmentation. Finally, specific product ions derived from the targeted precursor ions are selected for detection in the third quadrupole (Q3). Q3 is, generally, a low-resolution mass analyser that cannot transmit ions with isolation widths greater than 0.7–1.0 Da, without losing sensitivity. MRM detects transitions (Q1/Q3 MRM ion pairs) by utilising the unique features of a QqQ or QTRAP instrument
[21][110].
PRM is a newer targeted acquisition method and uses high-resolution accurate mass (HRAM) analysers, such as Orbitraps. In contrast to the QqQ-dependent MRM methods described above, Q3 is replaced with an HRAM mass analyser, enabling the parallel detection of all productions from targeted precursor ions, rather than selecting a limited number of productions (usually three transitions). PRM generates highly specific spectra for all productions derived from selected precursor ions, allowing for high-confidence targeted peptide identification. Since it uses several transitions to identify and quantify peptides, the predetermination of transitions and collision energy, which is required for MRM, is not required, reducing method development time. As opposed to MRM, PRM provides a significant improvement in signal-to-noise, while maintaining high sensitivity
[22][111].
Targeted proteomics can be used to bridge the chasm between biomarker candidate discovery and clinical utility. They allow us to look at the complete proteome, or a sub-proteome at the same time, to see whether there are any links between protein expression (or alterations) and disease development. One of the most promising areas is the analysis of distinct protein patterns linked with ovarian cancer as a discovery technique for prospective biomarkers. The tumour–host communication system is directly influenced by the proteomic tissue microenvironment, making it a viable source for biomarkers. Given the time and money required to bring a medicine to market, the availability of biomarkers capable of detecting probable drug failures early in development is critical. Multiple biomarkers will be required for accurate screening and diagnosis. Understanding oncogenic signalling and generating biomarkers predictive of patient outcome in response to specific medication regimens is another aspect that has considerable promise for therapeutics. Proteomic study can help us understand and prevent adaptive response in cancer cells, such as epigenetic changes and protein network reorganization, through post-translational modifications. However, like all other proteomics, targeted quantitative proteomics has some limitations—the throughput is constricted at around 50–100 proteins per analysis, and it has a low sample multiplexing ability
[23][57].
2.2. Peptidomics
Peptidomics is a new sub-division of proteomics and can, also, be used to shed light on new biomarkers. It is the study of peptides used to determine the exact form of each peptide. Like proteomics, it is used to identify new peptides that exist within tissue. It utilises quantification techniques to measure the relative level of peptides in varying environments.
Several studies have been done on peptides in oncology. Villanueva et al. communicated the differences between serum peptides in patients diagnosed with cancer and control experiments
[24][112]. Lopez et al. discovered a panel of serum peptides that distinguished stage OC cancer from controls
[25][113]. Fredolini et al. outlined 59 serum peptidome markers that are differentially expressed in OC versus benign conditions
[26][114]. Bery et al. relayed the peptide markers that are expressed in the ascites fluid of patients with OC against controls
[27][115].
Since proteolytic processes are deeply rooted in oncogenesis, peptidomics is expected to grow in the coming years with more advances in technology and understanding.
2.3. Exosomes
Exosomes, which are secreted by different cell types and are as small as 30–150 nm vesicles, play a critical role in intercellular communication. They have emerged as a compelling diagnostic and prognostic biomarkers for OC, as they may transport some tumour-associated proteins
[28][46]. These organelles can be found in various body fluids, such as urine, blood, CSF, breast milk, and saliva, making them more ideal, due to their being less invasive.
Exosome research provides large-scale protein analysis when using MS-based proteomics. For example, Sinha et al. used an MS-based technique to characterise isolated exosome proteomes of OC cell lines
[29][116]. However, a more recent study by Zhang et al. was the first-time exosomes were extracted from patient serum using LC-MS/MS and tandem mass tagging (TMT)
[30][117]. This study used enriched exosomes from plasma and characterised them using technologies such as dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), Western blot analysis, and transmission electron microscopy (TEM). The exosomal marker proteins CD81 and TSG101 were noted to be succinctly stained in the exosome samples, whereas the endoplasmic reticulum protein calnexin was not. Through this process, a net total of 294 exosomal proteins were identified. Two hundred and twenty-five of these proteins were found to be present in both the cancerous and non-cancerous samples. Exosomes isolated from the serum of women with various stages of OC were found to have higher levels of expression of 8 miRNAs (miRNA-21, miRNA-141, miRNA-200a, miRNA-200b, miRNA-200c, miRNA-203, miRNA-205, and miRNA-214) than those isolated from the serum of women with benign diseases, implying that miRNAs of circulating tumour exosomes can potentially be alternative biofluid biomarkers. Eventually, four genes (
LBP,
FGG,
FGA, and
GSN) were selected to be potential diagnostic and prognostic biomarkers because of their involvement in the apoptosis and coagulation pathways. The proteomic study found that
FGA and
GSN protein levels were upregulated in patients with OC. However, on the other hand,
FGG and
LBP were shown to be downregulated. It was, then, observed that high miRNA expression of
FGG or
LBP was associated with a shorter progression-free survival, indicating a poor prognosis for patients with epithelial cancer. Interactions between differentially expressed genes (DEGs) were noted using gene ontology analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and the protein–protein interaction network. This showed that hypercoagulation in epithelial OC is linked to the DEGs. The result of this study was the identification of four diagnostic and two potential prognostic biomarkers.
3. Proteomics in the Treatment of Ovarian Cancer
Whilst most proteomic research focuses on the diagnosis and prognostic indicators for ovarian cancers, the discovery of these new biomarkers presents an exciting opportunity in the treatment and management of OC.
3.1. Tackling Chemotherapy Resistance
Within the UK, the standard treatment for patients with OC involves debulking surgery followed by chemotherapy, namely carboplatin and paclitaxel. However, tumour resistance to platinum-based chemotherapy presents an emerging challenge to current regimes
[31][118]. Several groups have identified ways in which resistance occurs, many of which are related to key oncogenic signaling pathways
[32][119].
For example, disruption of the normal functioning of the PI3K/Akt/mTOR and MAPK pathway has been linked to resistance to platinum-based chemotherapy
[31][33][118,120]. Furthermore, lower expression of the presumed tumour-suppression gene RBPMS causes resistance to chemotherapy.
Whilst the over or under expression of certain proteins may indicate reduced sensitivity to chemotherapy, emerging evidence shows that targeted treatment against the pathways conferring resistance may help to overcome it
[34][121].
For example, Lee et al. reported the involvement of CXCR4 in promoting cell dormancy in response to chemotherapy, but treatment with CXCR4 antagonists in combination with other chemotherapy agents was found to have therapeutic effects in promoting cancer cell death. Zhang et al. identified several SENP1 inhibitors that inactivate the SENP1/JAK2/STAT pathway to overcome chemotherapy resistance
[35][122].
Table 1 summarizes biomarkers for drug resistance in OC, discovered by proteomic techniques.
Table 1.
Drug resistance markers.
3.2. Targeted Therapy Using Proteomics
The Cancer Genome Atlas and the International Cancer Genome Consortium have sequenced thousands of ovarian tumour specimens, which has resulted in the identification of novel genomic sequences that could be targets for therapeutic interventions
[37][123]. Whilst much work is underway to translate this into clinical practice, the two most evidenced therapeutic agents, which are FDA license approved, are poly (ADP-ribose) polymerase (PARP) inhibitors and VEGF/VEGF receptor inhibitors
[38][124].