Label-free methods for studying protein-protein interactions

Proteins are the building blocks of life, and mainly perform their roles through protein-protein interactions (PPIs). Thus, PPIs are essential to the normal function of the cells and the body, and disturbances in these interactions are an underlying cause for many diseases. Because PPIs are so vital, multiple methods have been developed to study these interactions. Typically, the methods are based on reporter molecules, although label conjugation may disrupt PPIs. Several label-free methods have also been introduced, which can be categorized into surface-based and solutionbased approaches. Surface-based methods are often very sensitive, but they require protein conjugation to a solid surface. Solution-based methods, on the other hand, are fully conjugation-free but suffer from low sensitivity. In this thesis, a label-free, solution-based Protein-Probe method with nanomolar sensitivity was developed for studying protein thermal stability and interactions. The method is based on an external probe peptide, the Eu-probe, and it does not require conjugation to the interacting proteins, avoiding potential disruption of the reactions. The Eu-probe does not significantly bind to native, intact proteins, and the timeresolved luminescence signal of the free probe is quenched by a modulator in the Protein-Probe solution. When denaturation reveals hydrophobic amino acids or binding events increase the overall surface area, the change in the protein structure enables the Eu-probe binding. Binding to the proteins protects the probe from the modulator and leads to a signal increase. The Protein-Probe was first applied to studying protein-ligand interactions and PPIs by observing their effect on protein thermal stability in thermal shift assays. PPIs were also monitored based on interaction-induced signal increase at elevated temperatures without a thermal shift, and in competitive thermal shift assays with small molecular ligands. The formation of large protein complexes and aggregates was successfully monitored at room temperature. The developed label-free Protein- Probe method has improved sensitivity over the current solution-based PPI detection techniques. The method enables monitoring different interaction types, as the external probe binds to a wide variety of proteins in multiple assay concepts. Thus, the Protein-Probe is a promising new method for PPI studies.Leimavapaat menetelmät proteiinien välisten vuorovaikutusten tutkimiseen Proteiinit ovat yksi elämän rakennuspalikoista, ja ne toimivat pääasiassa proteiinien välisten vuorovaikutusten (PPI:t) kautta. Tämän vuoksi PPI:t ovat elintärkeitä solujen ja kehon normaalille toiminnalle, ja häiriöt vuorovaikututuksissa ovat usean sairauden takana. Koska PPI:t ovat niin keskeisiä, niiden tutkimiseen on kehitetty useita menetelmiä. Tyypillisesti menetelmät perustuvat reportterimolekyyleihin, vaikka niiden kiinnitys saattaa häiritä PPI:tä. On olemassa myös useita leimavapaita menetelmiä, jotka voidaan jakaa pintapohjaisiin ja liuospohjaisiin menetelmiin. Pintapohjaisilla menetelmillä on usein hyvä herkkyys, mutta niiden heikkous on että proteiini täytyy konjugoida kiinteään pintaan. Liuospohjaiset menetelmät taas eivät vaadi ollenkaan konjugaatiota, mutta kärsivät usein huonosta herkkyydestä. Tässä työssä kehitettiin leimavapaa, liuospohjainen ”Protein-Probe” menetelmä, jonka herkkyys on nanomolaarisella alueella. Menetelmä perustuu ulkoiseen koetinpeptidiin, Eu-koettimeen, joten tutkittavia proteiineja ei konjugoida. Eu-koetin ei sitoudu merkittävästi natiivirakenteisiin proteiineihin, ja Protein-Probe-liuoksessa oleva modulaattori sammuttaa vapaan koettimen aikaerotteisen luminesenssisignaalin. Muutokset proteiinien rakenteessa, kuten denaturaatiosta johtuva hydrofobisten aminohappojen paljastuminen ja vuorovaikutuksia seuraava pintaalan kasvu, mahdollistavat Eu-koettimen sitoutumisen. Sitoutuminen proteiineihin suojaa koetinta modulaattorilta ja johtaa näin signaalin kasvuun. Protein-Probe-menetelmällä tutkittiin proteiini-ligandi- ja proteiini-proteiinivuorovaikutuksia seuraamalla niiden vaikutusta proteiinien lämpöstabiilisuuteen ”thermal shift” määrityksissä. Vuorovaikutuksia havainnoitiin myös perustuen sitoutumisen aikaansaamaan signaalin nousuun korkeassa lämpötilassa, ja kilpailevissa thermal shift määrityksissä pienten ligandimolekyylien kanssa. Suurten proteiinikompleksien ja aggregaattien muodostuminen havaittiin huoneenlämmössä. Kehitetty leimavapaa Protein-Probe menetelmä on herkempi kuin nykyiset liuospohjaiset tekniikat, ja sillä on mahdollista tutkia erilaisia vuorovaikutuksia, koska ulkoinen koetin sitoutuu moneen eri proteiiniin useassa määritystyypissä. Protein-Probe-menetelmä on siis lupaava uusi menetelmä PPI-tutkimukseen

Sensitive, homogeneous, and label-free protein-probe assay for antibody aggregation and thermal stability studies

Protein aggregation is a spontaneous process affected by multiple external and internal properties, such as buffer composition and storage temperature. Aggregation of protein-based drugs can endanger patient safety due, for example, to increased immunogenicity. Aggregation can also inactivate protein drugs and prevent target engagement, and thus regulatory requirements are strict regarding drug stability monitoring during manufacturing and storage. Many of the current technologies for aggregation monitoring are time- and material-consuming and require specific instruments and expertise. These types of assays are not only expensive, but also unsuitable for larger sample panels. Here we report a label-free time-resolved luminescence-based method using an external Eu3+-conjugated probe for the simple and fast detection of protein stability and aggregation. We focused on monitoring the properties of IgG, which is a common format for biological drugs. The Protein-Probe assay enables IgG aggregation detection with a simple single-well mix-and-measure assay performed at room temperature. Further information can be obtained in a thermal ramping, where IgG thermal stability is monitored. We showed that with the Protein-Probe, trastuzumab aggregation was detected already after 18 hours of storage at 60 degrees C, 4 to 8 days earlier compared to SYPRO Orange- and UV250-based assays, respectively. The ultra-high sensitivity of less than 0.1% IgG aggregates enables the Protein-Probe to reduce assay time and material consumption compared to existing techniques

Sensitive Label-Free Thermal Stability Assay for Protein Denaturation and Protein-Ligand Interaction Studies

In modern biochemistry, protein stability and ligand interactions are of high interest. These properties are often studied with methods requiring labeled biomolecules, as the existing methods utilizing luminescent external probes suffer from low sensitivity. Currently available label-free technologies, e.g., thermal shift assays, circular dichroism, and differential scanning calorimetry, enable studies on protein unfolding and protein-ligand interactions (PLI). Unfortunately, the required micromolar protein concentration increases the costs and predisposes these methods for spontaneous protein aggregation. Here, we report a time-resolved luminescence method for protein unfolding and PLI detection with nanomolar sensitivity. The Protein-Probe method is based on highly luminescent europium chelate-conjugated probe, which is the key component in sensing the hydrophobic regions exposed to solution after protein unfolding. With the same Eu-probe, we also demonstrate ligand-interaction induced thermal stabilization with model proteins. The developed Protein-Probe method provides a sensitive approach overcoming the problems of the current label-free methodologies

Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability

Thermal unfolding methods are commonly used as a predictive technique by tracking the protein's physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a 'Gold Standard' for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins

Protease substrate‐independent universal assay for monitoring digestion of native unmodified proteins

Proteases are a group of enzymes with a catalytic function to hydrolyze peptide bonds of proteins. Proteases regulate the activity, signaling mechanism, fate, and localization of many proteins, and their dysregulation is associated with various pathological conditions. Proteases have been identified as biomarkers and potential therapeutic targets for multiple diseases, such as acquired immunodeficiency syndrome, cardiovascular diseases, osteoporosis, type 2 diabetes, and cancer, where they are essential to disease progression. Thus, protease inhibitors and inhibitor-like molecules are interesting drug candidates. To study proteases and their substrates and inhibitors, simple, rapid, and sensitive protease activity assays are needed. Existing fluorescence-based assays enable protease monitoring in a high-throughput compatible microtiter plate format, but the methods often rely on either molecular labeling or synthetic protease targets that only mimic the hydrolysis site of the true target proteins. Here, we present a homogenous, label-free, and time-resolved luminescence utilizing the protein-probe method to assay proteases with native and denatured substrates at nanomolar sensitivity. The developed protein-probe method is not restricted to any single protein or protein target class, enabling digestion and substrate fragmentation studies with the natural unmodified substrate proteins. The versatility of the assay for studying protease targets was shown by monitoring the digestion of a substrate panel with different proteases. These results indicate that the protein-probe method not only monitors the protease activity and inhibition, but also studies the substrate specificity of individual proteases.</p

Nanomolar Protein-Protein Interaction monitoring with a label-free protein-probe technique

Protein-protein interactions (PPIs) are an essential part of correct cellular functionality, making them increasingly interesting drug targets. While Förster resonance energy transfer-based methods have traditionally been widely used for PPI studies, label-free techniques have recently drawn significant attention. These methods are ideal for studying PPIs, most importantly as there is no need for labeling of either interaction partner, reducing potential interferences and overall costs. Already, several different label-free methods are available, such as differential scanning calorimetry and surface plasmon resonance, but these biophysical methods suffer from low to medium throughput, which reduces suitability for high-throughput screening (HTS) of PPI inhibitors. Differential scanning fluorimetry, utilizing external fluorescent probes, is an HTS compatible technique, but high protein concentration is needed for experiments. To improve the current concepts, we have developed a method based on time-resolved luminescence, enabling PPI monitoring even at low nanomolar protein concentrations. This method, called the protein probe technique, is based on a peptide conjugated with Eu chelate, and it has already been applied to monitor protein structural changes and small molecule interactions at elevated temperatures. Here, the applicability of the protein probe technique was demonstrated by monitoring single-protein pairing and multiprotein complexes at room and elevated temperatures. The concept functionality was proven by using both artificial and multiple natural protein pairs, such as KRAS and eIF4A together with their binding partners, and C-reactive protein in a complex with its antibody

Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability

Thermal unfolding methods are commonly used as a predictive technique by tracking the protein's physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a 'Gold Standard' for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins

Rapid high-throughput compatible label-free virus particle quantification method based on time-resolved luminescence

Viruses play a major role in modern society and create risks from global pandemics and bioterrorism to challenges in agriculture. Virus infectivity assays and genome copy number determination methods are often used to obtain information on virus preparations used in diagnostics and vaccine development. However, these methods do not provide information on virus particle count. Current methods to measure the number of viral particles are often cumbersome and require highly purified virus preparations and expensive instrumentation. To tackle these problems, we developed a simple and cost-effective time-resolved luminescence-based method for virus particle quantification. This mix-and-measure technique is based on the recognition of the virus particles by an external Eu3+-peptide probe, providing results on virus count in minutes. The method enables the detection of non-enveloped and enveloped viruses, having over tenfold higher detectability for enveloped, dynamic range from 5E6 to 3E10 vp/mL, than non-enveloped viruses. Multiple non-enveloped and enveloped viruses were used to demonstrate the functionality and robustness of the Protein-Probe method

Työkierto sosiaali- ja terveysalalla - kirjallisuuskatsaus

Tämän opinnäytetyön tarkoitus on kuvata työkiertoa sosiaali- ja terveysalalla, millaisia hyötyjä työkierrosta on ja millaisia haasteita se voi aiheuttaa. Tavoitteena tässä opinnäytetyössä oli kerätä suomenkielistä tietoa työkierrosta sosiaali- ja terveysalalla ja tiivistää se kirjallisuuskatsauksen muotoon. Opinnäytetyöhön valittiin kymmenen aiheeseen liittyvää suomenkielistä opinnäytetyötä, jotka toimivat aineistona tässä opinnäytetyössä. Muina lähteinä käytettiin erilaisia aiheeseen liittyviä julkaisuja. Työkierrolla tarkoitetaan siirtymistä ennalta sovituksi ajaksi omista työtehtävistä toiseen työyksikköön. Työkierron tavoitteena on muun muassa henkilökunnan työhön liittyvän tiedon lisääminen ja käytännön taitojen kehittäminen. Työkierto voidaan ajatella yhdenlaisena organisaation henkilöstön kehittämisen muotona. Opinnäytetyön tuloksista selvisi, että työkiertoa pidettiin positiivisena kokemuksena, se lisäsi hoitajien motivaatiota ja työssä jaksamista. Tärkeimmäksi asiaksi kuitenkin mainittiin hoitajan ammatillisen osaamisen kehittyminen työkierron aikana. Haasteiksi koettiin muun muassa huono tiedonkulku tai sen puute eri tahojen välillä sekä perehdytyksen ja esimiehen tuen puute ennen työkiertoa ja sen aikana. Opinnäytetyön tuloksia voidaan käyttää erilaisissa työkiertoa kehittävissä projekteissa ja työkiertoon perehdytettäessä. Niitä voivat hyödyntää myös yksittäiset henkilöt esimerkiksi tämän opinnäytetyön tekijä, sosiaali- ja terveydenhuollon ammattilaiset ja muuten aiheesta kiinnostuneet yksityishenkilöt. Jatkotutkimusaiheena voisi aihetta tutkia lisää työhyvinvoinnin näkökulmasta, esimerkiksi näkyykö työkierto hoitajien työhyvinvoinnissa työntekijän palattua takaisin omaan työyksikköön. Lisäksi voisi vertailla sosiaalialan ja hoitoalan työkierron eroavaisuuksia työkierron näkökulmasta. Mitkä keinot edistävät ja mitkä estävät työkiertoa.The purpose of this thesis was to describe the job rotation in the field of social and health care, what kind of benefits the job rotation offers and what kind of challenges it may cause. The aim of this thesis was to collect information in Finnish about the job rotation in the social and health care and summarize it as a literature review. Ten theses in Finnish related to this topic were chosen as material for this study. Furthermore, other topic related publications were used. Job rotation means the transition from one ́s own duties to another unit for a pre-agreed period of time. The aim of the job rotation is inter alia to increase the work related knowledge of the personnel and the development of the practical skills. Job rotation can be considered as one kind of a form of an organization ́s personnel development. The results showed that the job rotation was considered as a positive experience. Moreover, it increased the nurses ́ motivation and coping at work. However, the development of the professional skills of the nurses was mentioned as the most important issue. The challenges was inter alia insufficient communication or the lack of it between the parties. Furthermore, the lack of orientation and the support from the supervisor before the job rotation or during it were also found challenging. The results of this thesis can be used in different projects which focus on developing the job rotation and during the orientation of the job rotation. In addition the results can be applied by individuals, for instance, the author of the thesis, social and health care professionals and other individuals interested in the subject. As a follow-up research, this topic could be studied further from the point of view of well-being at work. The topic could be does the work rotation show nurse ́s well-being at work after the employee ́s return back to own work unit. In addition, the differences in job rotation between social and health care from the point of view of work rotation could be compared. Which methods enhance and which prevent job rotation

Design of neonatal dried blood spot immunoassays for two novel biomarkers

Spinal muscular atrophy (SMA) is a genetic disorder that leads to muscle weakness and atrophy and, in the most severe form, death before the age of two. It is caused by lack of survival of motor neuron (SMN) protein. Congenital heart defects are malformations caused by abnormal development before birth. They range from asymptomatic to fatal and can be detected by measuring the plasma concentration of N-terminal pro-B-type natriuretic peptide (NT-proBNP). The aim of this study was to develop immunometric immunoassays that could detect SMN and NT-proBNP from neonatal dried blood spot (DBS) samples using an automated laboratory analyzer, to detect, respectively, the SMA phenotype or congenital heart defects at an early stage. Capture antibodies were attached to a solid surface via adsorption coating or biotin-streptavidin interaction. Tracer antibodies were labeled with N1-Eu- or N3-Eu-chelate. From DBS samples, 3.2 mm disks were punched into the microtiter wells. The assays were performed either sequentially on different instruments or by the GSP instrument. SMN detection requires lysis of the blood cells. Using optimized lysis and assay protocols, the SMN concentration measured from DBS samples of healthy individuals was at most 4.3 ng/ml, whereas a concentration range of 8.1-11 ng/ml was acquired from whole blood samples. The assay was functional but lacks sensitivity for detecting the SMN levels of SMA-positive DBS samples. NT-proBNP was assayed from DBS samples with sufficient sensitivity, and the assay was transferred to the GSP instrument. The NT-proBNP concentrations of 83 neonatal DBS samples were measured to be 580-10 800 pg/ml and fitted a log-normal distribution. Clinical samples would be required to evaluate the diagnostic value of this assay