Phosphoproteomics as a Promising Tool for ...

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Phosphoproteomics as a Promising Tool for Broadening the Analysis of Clinical Sampies and for the Fight Against Cancer Disease Natalia Miękus and Tomasz Bączek*

Department of Pharmaceutical Chemistry, Medical Universily of Gdańsk, Hallera 107, 80-416 Gdańsk, Poland Abstraet: Proteornic approaches posses a huge potential in understanding diseases such as cancer. These analyses rely on the extraction of proteins frorn clinical specimens and their subsequent enzymarie digestion in peptides prior to mass spectrometric analysis. The characterisation of chosen subproteomes - like phosphoproteorne - which are rich reservoirs of potential biomarkers for disease, requires much more sophisticated procedures due to the additional phosphoproteornic sample preparation steps. The addition of sophisticated analytical procedures is mandatory but make the parallel processing of clinical samples prone to plenty of errors and, thus compromise the reproducibility needed for confident comparative high-throughput studies, The presented review focuses on relevant developments in the phosphoproteomic field that could, in the near futurę, facilitate the rapid discovery of tumour biomarkers or new drug targets for cancer treatment. 1n terms of methodological advances, the enrichment ofphosphoproteins and mass spectrometry detection ofpeptides will be especially highlighted. The discussion part will also eontributę to new areas of interest for scientists that are investigating phosphoproteins.

Keywords:

Cancer,

enrichment

techniques,

phosphopeptides,

l. INTRODUCTlON Clinical proteornics, as a field of science the main goal of which is to analyze the total protein composition (proteorne) of an organism, tissue or celi and further use the gained data for medical purposes, is gaining progressively more attention. Among others, scientists that are dealing with serious diseases like cancer, are truły enthusiastic about proteomics and the undoubted importance of this approach in the fight against life-threatening diseases. Obtaining a profound knowledge of proteins - their structure, localization, function and mutual interactions - is a key factor in revealing the eontribution of proteins in physiological and pathophysiological processes in the human body [1, 2). Current developments in analytical and bioinforrnatic tools in proteomics have led to relevant advances in the diagnosis, monitoring, as well as the treatment of different types of cancer [3-5). Proteomics is considered to be ahead of other -omics fields of science like genornics [6). This is caused by the fact that physiological changes within the celi may be modulated by changes in protein levels as well as by protein post-translational modifications (PTMs), which are not detectable while conducting gen e expression profiling, as in genomics studies [7]. Nevertheless, proteome is constantly being subjected to dynamie changes, Iike the abovementioned PTMs. Therefore, the identification of clinically relevant proteins, for exarnple biomarkers for the early detection of cancer, in highly complex protein mixtures (corresponding to materials *Address correspondence to this author at the Departmcnt of Pharmaceutical Chcmistry, Medical University of Gdańsk, Hallera 107,80-416 Gdańsk, Poland; Tel: +48 58 349 16 35; Fax: (48) (58) 349 16 30; E-mail: [email protected]

l 875-616X/l 4 $58.00+.00

phosphoproteomics,

prefractionation,

tandem

mass spectrometry.

that are urgent to analyze in clinical proteornics - human biological fluids - blood, urine, saliva - tissues and celi cultures) is troublesome [8). Thus, a trend to narrow the area of potential cancer biomarkers or the identification ofnew drug targets of subproteornes that are relevant for the clinician, is receiving increasing attention nowadays [9, 10). Among 300 PTMs already described in literature, the phosphorylation of proteins plays a key role in celI metabclism, division, signal transduction, homeostasis, regulation processes or localization. Phosphorylation can occur on approximately 30% of proteins in a celi during its lifecycle [11, 12). Moreover, unbalanced phosphorylation is known to promote numerous life-threatening diseases such as cancer [13-15]. Cornparative phosphoproteomics can distinguish smali but important changes in modifications in protein structure, thus facilitating drug target protein identification or revealing potential cancer biomarkers [16). Phosphoproteomics is a subfield of proteornics that investigates phosphoproteome. Examining that particular subproteorne is ratber intricate since genes do not code PTMs like phosphorylation. Therefore, it carmot be predicted in a straightforward manner. Furthermore, phosphorylation is a transicnt, reversible, dynarnic modification, with low stoichiornetry, and one protein can undergo this PTM on different amino acids (the most often on serine, tyrosine and/or tryptophan) within its sequence. Moreover, phosphoproteins are less abundant that their non-phosphorylated counterparts. Studies of phosphoproteins are recciving more and more attention, nevertheless, only 2% of phosphorylation sites of proteins have been characterized so far [7, 17]. There is a need to discover and point out further modification sites and to eontinue the success of tyrosine kinase-based cancer therapeutics, for instance [18). © 2014 Bentharn Science Publishers

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Considering all the above, there is a need to earry out phosphoproteomie experiments using highly developed technologies, sophisticated techniques for the fractionation of the sample and special bioinformatie tools for experimental data analysis. The presented review will foeus on major developments in phosphoproteomie approaehes that faeilitate progress in the fight against cancer disease. Attention will be put on enrichment methods, which are most important for pro per phosphoproteome investigation and the further identification of phosphoproteins in complex sam pIes. 2. EXPERIMENT

AL WORKFLOW

2.1. Phosphoproteome

- MAJOR

STEPS

Enrichment

Normally, global studies of phosphoproteins require sophistieated methods for sample preparation before analysis, mainly due to the high background of non-phosphorylated peptides and the low stoichiometry of phosphopeptides in a trypsin digest. Therefore, enriehment of the chosen subproteorne prior to LC-MS/MS (liquid chromatography tandem mass spectrametrys analysis is required to obtain optima l phosphoproteome coverage. Numerous phosphopeptide enrichment approaches have been established [19). Among them are: imrnunoprecipitation with specific antibodies [20], enrichment with strong cation exchange chromatography, immobilized metal ion affinity chromatography (\MAC) [21], metal oxides/hydroxides su ch as titanium dioxide (Ti02) [22] or sequential elution from [MAC [23]. Ti02 and IMAC are most frequently used. Both have their advantages and disadvantages, but Ti02 is appreciated for its robustness and higher tolerance towards salts and detergents than IMAC. One of the problems associated with the lMAC technique is a high level of nonspecific binding when used for phosphopeptide enrichment of highly complex peptide sampIes [13]. On the other hand, Ti02 has a very strong affinity to bind multi-phosphorylated peptides, and their elution fTOm Ti02 beads is often not complete. Therefore, much effort has been made to design a resin with the advantages of both techniques: lMAC and Ti02, but without their limitations. Recently, a new matrix has been proposed [24). It is composed of Ti4+ ions binding to lMAC resin. Owing to its high resistance towards salts and detergents, high specificity for phosphopeptides (as Ti02 resin), better recovery of mu Itiphosphorylated peptides and comparable recovery of monophosphorylated peptides in comparison with Ti02, and the low amount of starting material necessary to process for enrichment (good results for I ug of a not complex sarnple), the TiHf IMAC resin is receiving more at1ention in phosphoprotcome studies.

2.2. Phosphopeptide

Detection

Several analytical techniques exist for the detecrion of phosphorylation. Among them mass spectrometry (MS) is mostly applied. This is on account of its sensitivity and specificity, as well as the possibility of high-throughput identification and the quantifieation of proteins present in complex biological mixtures [II). MS preceded by high performance liquid chromatography (HPLC) fractionation of the sample is frequently used [25]. This approach facilitates the identification of single phosphoproteins in highly complicated matrices and reduces the suppression of phosphory-

lated peptides in mass spectra. Mass spectrometry has aIs o an additional advantage over other analytical methods i.e. phospho-specific antibodies, because here sequences of phosphorylated proteins can be unknown prior to analysis [26]. Therefore, it is a perfect match for biornarker studies, where new, often small-size phosphoproteins need to be found among numerous interfering compounds presented in the sarnple [27]. Nevertheless, only a fraction of proteome proteins are phosphorylated at any given time and the phosphoproteins are less abundant than their non-phosphorylated counterparts. Moreover, predominant phosphoproteomic mass spectrometry analyses rely on a positive ion ionization mode. In this condition, the ionization 01' phosphopeptides is much lower than the ionization of non-phosphorylated peptides. Furtherrnore, collisionally induced dissociation (CTD) fragmentation is usually employed. After CID, the mass spectra of phosphopeptides are mostly dominated by highly abundant peaks arising from the neutrai loss of the phosphate group r261. Therefore, the visualization of backbone fragmentation, which is essential to determine the amino ac id sequence, is often hindered. Some of the abovementioned complications can easily be overcome by the use of sophisticated enrichment and fractionation methods prim to MS analysis. Moreover, the application of multistage activation (MSA) Ol' neutrał loss triggered MS3 (MS/MS/MS) modes (Fig. l b 1 and lb2) could be supportive to force the neutraJ loss peak dominance in phosphopeptide spectra [26,28]. In those techniques, the mass-tocharge ratio (m/z) and the intensity of the intact peptide precursors are recorded by an initial MS scan - known as fuli scan MS. Then, the m/z values for peaks (list of masses) with high intensity are automatically selected for sequencing by tandem MS (MS/MS) (Fig. 1a). The two first steps are equivalent to the usually used MS/MS mode, but in MSA or MS3 mode there is an additional third step. lt relies on the fragmentation of the neutral loss peak and, therefore, the excess of energy is postponed for backbone cleavages (Fig. .1 b). Here, the visualization of backbone fragment peaks is promoted. Nowadays, high-resolution mass spectrometers prefcrably used in phosphoproteomics, mak e multistage activation not obligatory to apply. In cancer studies the most urgcnt is the analysis time, Therefore, the use of the MS/MS mode together with highly advanced instrumentation, which is much fas ter than the multistage activation mode, is sufficient [29]. Applications of alternative fragmentation techniques to ClD, such as electron capture dissociation (ECO) Ol' electron transfer dissociation (ETO) [13,30], could also hamper neutral loss peak superiority in the peptide spectrum. ECO fragmentarion allows the retention of the phosphate group on the amino acid. Therefore, information on both: the position of phosphorylation and the amino acid sequence 01' phosphopeptides, could be gained. Owing to ECO also peptides with masses >2500 Da and charges greater than +2 are more likely to be detected than after CID fragmentarion. ECO and ETO methods of fragmentation are classed as powerful tools in phosphoproteornics, but still need to be optimized [31].

Current Pharmuceutical Analysis, 2014, Vol. J O,No. 3

Phosphoproteomics as a Promisil/g Tool for Brnadening the Anałysis

a) lons inlet

----1'--

MSl

._o_•....H

Collision celi H~e

He

MS2

H__

Collislon celi

MSl b) lons inlet

H,He

225

MS2

bl

b2

Collision celi H. He

He He He

Neutral loss

MS2 Neutral loss Iragmcnts

Fig. (1). rIlustration of MS/MS (a) and MS3 (b: b 1; b2) modes.

COMMON AND UNEXPECTED EST INPHOSPHOPROTEOMlCS

FIELD S OF JNTER-

Nowadays, after a great improvement in the development of phosphoproteomic methodology, there is a tendency to explore phosphoproteomics as an alternative in cancer disorders or even in distinct organisms other tban humans or ani-

mals. lt is worth mentioning the use 01' phosphoproteomic studies in cardiac diseases [32, 33]. A deregulation of balance between kinases and phosphates was discovered in many cardiac disorders. This revelation could be helpful in the field of cancer. Detected phosphoproteins or valid signalling networks could serve as new potential tberapeutic targets. Already pbosphoproteomic approaches facilitate the development of useful kinase modulators, which have emerged as an important class of cardiovascular drugs. Moreover, the phospho-research of neurodegenerative disorders such as Alzheimer's disease (AD), revealed inadequate phosphorylation of some important proteins - like the peptidyl-prolyl cis/trans isomerase Pin I. lt was assumed that Pin l displays several post-translational modifications, which are specific in tauopathies - like AD - and may be useful as biomarkers [34]. Furthermore, anticoagulant therapies needed phosphoproteomics to gcnerate novel, more efficient drugs with a lack of serious side effects, So far, discovered inhibitors of the protease-activated receptor (P AR) l - which is the main target of anticoagulant therapies nowadays - increase the risk of bleeding in patients. Phosphoproteomic studies which aim to reveal new, interesting candidates for further studies in terrns of new anticoagulant therapies are being conducted [35]. As regards phosphoproteomics in non-human research, the phosphoproteomes of several plants we re analyzed [36, 37]. The importance of phosphoproteins in the photoresponses of plants was studied. The specific control of proteins by reversible phosphorylation in the flagellurn was discovered as well. The f1agellum was analyzed due to its usefulness in celi locomotion and its widespread occurrence in nature: in prokaryotic as well as eukaryotic cells.

Last but not least, following the well-known fact that som e bacteria genes encoded enzymes similar to those in eukaryotes, the study of bacteria phosphoproteome was carried out. As an example, the Streptococcus pneumoniae is presented here. Its gene sequencing revealed the presence of a gene coded as a eukaryotic-type Serine/Threonin protein kinase. lt was demonstrated that the discovered gene is essential in bacteria for successful lung infection and blood invasion in mice and plays an important role in the celi division cyc\e 01' S. Pneumonia [38]. To sum up, phosphoproteomics is used in a wide range of areas and could be a field of interest for scientists that so far have not used this approach in their studies. Phosphoproteomics could open a new door to facilitate discoveries in various disturbances or diseases, not only in humans but also in other species.

DISCUSSION AND CONCLUSION Currently, the Human Proteome Organization (HUPO) continuing the successfully accomplished Human Genome Project (HUGO) - has started a worldwidc project which aims at analysing the entire human proteome (Rumem Proteome Project, HPP) [39, 40]. For this purpose, numerous proteomic researches, which are investigating varied types of biological sampies, are being conducted. Scientists focus also on chosen subproteomes such as phospho- or glycoproteome [15, 41, 42]. Clinical PTM rcscarch studies, couplcd with bioinformatic tools, are routinely carried out with growing success. Phosphoproteomes, especially rich in potential diagnostic biomarkers, are enthusiastically investigated. Recent developments in phosphoproteomics have contributed to a deeper examination of the cancer phosphoproteome (Table 1). The growing interest in phosphoproteomics is well documented in the aspects of drug discovery, target identification, revelation of signalling network s within the celi and/ar between cells, checking the toxicity of the drugs, biomarker discovery and the profil ing of patients' body fluids [43]. Still, further improvement in the preparation of phospho-enriched sampies and the detection of single phosphoproteins from complex protein mixtures (as biological sampies originating frorn patients suffering from turnours) is necessary [44,45].

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Table 1.

Detalled Importance of phosphoproteomics

Miękus and Bączek

for the treatrnent

Type of cancer

Ol"

Importance

monitoring of various cancers.

of Phosphoproteomics

Source

Cancers in general

Oncogenic kinase signaling study, new drug target discovcries

[43,54,55]

Triple Negative Breast caneer (TNBC)

Treatment ofTNBC

[56,57J

Breast cancers, Gastrointestinal stroma I tumors (G 1ST)

Revealing the causes of drug resistance, searehing for more appropriate therapies

[52,58-63]

Lung eaneer, Glioblastoma, Gastrointestinal Stromal Tumors (GIST)

Novcl oneogon ie targets

[48,50,62,64]

Leukemia

Kinase inhibitors

(65,66]

Human epithelial tumors

Haematologieal Tumors

Study of thc epiderrnal

growth faotor (EGF)-like proteins tor new therapeutic oportunities

ldentitication ofnovel markers ofresistance or sensitivity to drugs, revealing the role ofkinanses in cancer development

Unquestioned advances in analytical techniques, apparatus and tools for data analysis have led to the growth in the usefulness of the phosphoproteomic approach in revealing the human phosphoproteome during health and disease [46, 47-49]. In-depth investigation of the role of different kinases and phosphatases in the initiation or progression of carcinogenesis and the search for phosphoproteins that are differently secreted during cancer development, facilitates new insights in this serious disease. The growing investigation of enzymes responsibJe for phosphorylation allows new discoveries in the field of kinases that are incorrectly secreted only by tumour cells. The improper activation of tyrosine kinases represents an important oncogenic mechanism and is being investigated for example for glioblastoma [50]. Those revelations are folIowed by the development of new kinase inhibitors or new therapeutic targets for cancer. Kinase inhibitors are now among the new promising anti-cancer drugs aJready in use for cancer patients as well as in the c1inical triais phase [46, 51]. StiJJ, most solid tumors and the study 01' tyrosine kinases that drive disease, remain unknown, limiting our ability to identify drug targets and predict rcsponses [18].

[49,68]

That is why advances in phosphoproteomic tools focused on cancer research are being wideIy explored and tested. What is also worth mentioning is the use of phosphoproteomic data to distinguish various cancers. Owing to the profound investigation of phosphoproteomes of different haematologicaJ cancers (myeJoid leukaemia, Iymphoma, multiple myeloma), a c1assification according to tumour type was compiled and the sensitivity to kinase inhibitors was described, It was revealed that unbalanced phosphorylation in investigated eancer cells correlates with tumour sensitivity and the response to kin as e inhibitors used for the treatment. This could be a step towards improvement in personalized therapy [49). Moreover, the study of phosphoproteins revealed the resistance of some sub-types of the cancers to the therapy. For instance, the majority of breast cancers are dependent on estrogen (ER) receptors. Therefore, drugs like tamoxifen are used for the therapy of this cancer. Nevertheless, after some time, 30 % of ER-dependent breast cancer patients becorne resistant to the therapy. Profound phosphoproteomic studies

Identification of unba.lanced signaling patterns in tumor celi s

Biomarkers for detection and monitoring of cancer

New knowledge of carcinogenesis mechanism

Fig. (2). Phosphoprotcomics impact in fight against canccr.

[67J

Current Pharmuceutical Anulysis, 2014, Vol. J O, No. 3

Phosphoproteomics as a Promising Tool for Bromlening the Analysis

were conducted to reveal if there is any disturbance on the level of phosphoproteins between those breast cancers. Studies confirmed that careful research of the phosphorylation pattern among bre as t cancer patients should be continued since the data showed that there are differences in phospboproteornes between breast cancer patients [52]. Obviously, prevention is the major issue in the anticancer approach. Therefore, the early detection of cancer, in the phase when a cornplete cure of the patient could be achieved, needs to be improved. Clinical phosphoproteomics focuses on the analysis of phosphoproteomes of easily accessible biological fluids obtained from cancer patients and the detection of differentiaUy secreted phosphoproteins that appear onI y during carcinogenesis. Subsequently, the detected phosphoproteins can be classed as potential cancer biom arkers for the detection and monitoring of disease [27] (Fig. 2). The study of phosphoproteins as potential biomarkers of disease is still a complicated task; nevertheless, in the near future its irnportance in this aspect of cancer treatment and detection will grow owing to improvements in sample preparation and experirnental workflow design [53]. Nowadays, the rapid growth in interest towards the cancer phosphoproteome has led to important findings in the field of cancer. Gaining profound knowledge about that subproteome, rich in clinically important proteins, facilitates the revelation of possible carcinogenesis mechanisms and patterns that are worth concentrating on while searching for new anti-cancer targets, therapies or cancer biomarkers. Ultimately, for the final result of the phosphoproteomic fight against cancer, the world needs to wait alittle longer. CO

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FLlCT OF INTEREST

The authors eonfirm that the eontent of this article has no conflict of interest.

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ACKNOWLEDGEMENTS This Project was supported by the Ministry of Science and Higher Education of the Republic of Poland, from the quality-prornoting subsidy, under the Leading National Research Centre (KNOW) programme for the years 2012-2017. REFERE [I]

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Lagadinou, E.D.; Sach, A.; Cal1ahan, K.; Rossi, R.M.; Neering, SJ.; Minhajuddin, M.; Ashton, J.M.; Pei, S.; Grose, V.; O'Dwyer, K.M.; Liesveld, J.L.; Brookes, P.S.; Becker, M.W.; Jordan, C.T. BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stern cel1s. Celi Stem Celi, 2013,12(3),329-341. Yarden, Y. The EGFR family and its ligands in human cancer. signalling mechanisrns and therapcutic opportunities, Eur. J. Car/cer, 2001,37 Suppl 4, 3-8. Piazza, F.; Manni, S.; Ruzzene, M.; Pinna, L.A.; Gurrieri, C.; Semenzato, G. Protein kinase CK2 in hematologie malignancies: reliance on a pivotal cel1 survival regulator by oncogenic signaling pathways. Leukemia, 2012, 26(6), I 174-1179.

Accepted:

April 03, 2014