Development of liquid chromatography mass spectrometric methods for quantification of metabolites from cellular level to clinical biomarkers

Metabolites are low molecular weight compounds participating in different functions of cellular systems. Metabolites can be used as diagnostic biomarkers for numerous diseases. Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a powerful tool in quantification of metabolites from various sample matrices. Good sensitivity and specificity are the main benefits of the technique. Mass spectrometry is commonly used in industry, drug research and clinical diagnostics. Extensive validation of newly developed analytical methods will construct the basis to a reliable assay, and it is significant especially when analysing e.g. patient samples. The aim of this study was to develop quantitative assays for metabolites from biological samples for biomedical research and clinical diagnostics. We designed and constructed an on-line high performance liquid chromatography (HPLC) equipment and validated an assay for direct quantification of extracellular metabolites from cell cultivation. Automated sampling for LC-MS/MS analysis of intracellular metabolites was connected to the on-line system. The on-line analysis improves the methodology and shortens the time of analysis. Furthermore, a frequent sampling data can provide valuable information about physiological indications in various cell cultivations. On-line HPLC is suitable for various biotechnological applications because of its ability to monitor and collect data during cell cultivation. We developed and validated LC-MS/MS assays for neuroendocrine tumor (NET) biomarkers 5-hydroxyindole acetic acid (5-HIAA) and vanillylmandelic acid (VMA) from human serum. Generally, urinary HPLC assays are used for the determination of NET markers. HPLC assays have certain limitations and 24-h urine collection is laborious. Our LC-MS/MS assays are specific, fast and well suited for diagnostics of NETs. Furthermore, guidelines for urine collection advise to refrain from serotonin-containing foods for three days before sample collection. We showed that such a diet restriction before serum 5-HIAA assay is not necessary. Instead, one day serotonin-free diet before sampling is sufficient because the half-life of 5-HIAA in circulation was found to be 1.3 hours. All assays developed during this study were sensitive and had a wide linear range. Our serum 5-HIAA LC-MS/MS assay is routinely used for the analysis of NET patient samples at the Helsinki University Central Hospital Laboratory, HUSLAB. Serum VMA LC-MS/MS assay will be in routine use in the HUSLAB in near future. Furthermore, On-line HPLC Ltd, (Helsinki, Finland) has commercialized the on-line HPLC equipment developed in this study.Metaboliitit ovat pienen molekyylipainon omaavia yhdisteitä, jotka osallistuvat erilaisiin toimintoihin solujen aineenvaihdunnassa. Eri metaboliitit toimivat myös useiden sairauksien merkkiaineina ja niiden pitoisuuden mittausta käytetään apuna sairauksien toteamisessa sekä seurannassa. Nestekromatografia (LC) yhdistettynä massaspektrometriaan (MS) on tehokas analyysitekniikka metaboliittien määritykseen. LC-MS -menetelmiä käytetään laajalti esimerkiksi teollisuudessa, lääketutkimuksessa sekä kliinisessä diagnostiikassa. Menetelmän validointi eli toimivuuden testaus on erittäin tärkeä vaihe uusien määritysmenetelmien kehityksessä. Validointi takaa, että menetelmä toimii halutulla tavalla ja että analyysitulokset ovat luotettavia, mikä on erityisen tärkeää varsinkin analysoitaessa potilasnäytteitä. Tämän työn tarkoituksena oli kehittää ja validoida kvantitatiivisia määritysmenetelmiä metaboliiteille käyttäen hyödyksi LC-MS -tekniikoita. Kyseisiä menetelmiä voidaan hyödyntää biolääketieteellisessä tutkimuksessa ja kliinisessä diagnostiikassa. Suunnittelimme ja valmistimme korkean erotuskyvyn nestekromatografia (HPLC) -laitteiston ja validoimme menetelmän solunulkoisten metaboliittien reaaliaikaiseen määritykseen suoraan solukasvatuksesta. Lisäksi keräsimme näytteitä solukasvatuksesta automaattisella näytteenottolaitteistolla ja määritimme kerätyistä näytteistä solun sisäisiä metaboliitteja LC-MS/MS -menetelmällä. Reaaliaikaisen mittauksen avulla saatiin lisää hyödyllistä tietoa solujen aineenvaihdunnasta. Metaboliitit 5-hydroksyyli-indoliasetaatti (5-HIAA) ja metoksihydroksimandelaatti (MOMA, VMA) ovat neuroendokriinisten kasvaimien merkkiaineita. Yleensä näitä merkkiaineita on määritetty potilaan vuorokausivirtsasta HPLC -menetelmällä yhdistettynä elektrokemialliseen tai flurometriseen detektioon. Nämä menetelmät ovat kuitenkin alttiita häiritseville yhdisteille, kuten lääkeaineille tai toisille metaboliiteille. Lisäksi vuorokausivirtsan keräys ja esikäsittely on hankalaa ja aikaa vievää sekä potilaalle että laboratoriolle. Näiden syiden vuoksi kehitimme ja validoimme suoraviivaiset LC-MS/MS -menetelmät 5-HIAA:n ja VMA:n nopeampaan ja luotettavampaan määritykseen verinäytteestä. Kehitetyt menetelmät ovat spesifisiä, nopeita ja sopivat hyvin kliiniseen diagnostiikkaan. Aiemmat potilasohjeet neuvoivat välttämään tiettyjä ruoka-aineita kolme päivää ennen 5-HIAA virtsanäytteenottoa. Me osoitimme, että yksi päivä riittää kyseisten ruoka-aineiden välttämiseen ennen verinäytteenottoa. Kaikki tässä työssä kehitetyt määritysmenetelmät olivat herkkiä, nopeita sekä paransivat analytiikkaa. Seerumin 5-HIAA -menetelmä on ollut rutiinikäytössä Helsingin yliopistollisen keskussairaalan laboratoriossa, HUSLAB:ssa vuodesta 2013 ja seerumin VMA -menetelmä otetaan käyttöön lähiaikoina. Lisäksi On-line HPLC -niminen yritys on kaupallistanut tässä työssä kehitetyn HPLC-laitteiston

Label-free mass spectrometry proteome quantification of human embryonic kidney cells following 24 hours of sialic acid overproduction

Peer reviewe

Plasma bradykinin concentrations during septic shock determined by a novel LC-MS/MS assay

Background: Bradykinin is an important mediator of inflammation and vascular permeability and could have an important role in the development of septic shock. Measurement of bradykinin by immunological methods may suffer from interference and lack of specificity. We developed and validated a liquid chromatography mass spectrometry assay (LC-MS/MS) for plasma bradykinin. Methods: We used plasma samples from healthy volunteers (n = 19) and patients with septic shock (n = 47). Stable isotope bradykinin internal standard was added to samples before solid-phase extraction and quantification by LC-MS/MS. Stability of bradykinin was studied for 12 months. Results: Our assay has good sensitivity (0.1 nmol/l) and a wide linear range (0.1-1000 nmol/1). Bradykinin added to plasma was stable for 12 months at -20 degrees C when a mixture of protease inhibitors was added at sampling but degraded during repeated freezing and thawing. Bradykinin concentration in plasma from septic shock patients (<0.1-0.6 nmol/l) did not change significantly during shock and recovery but differed slightly from that in healthy individuals (0.5-1.1 nmol/1). Conclusions: Our bradykinin assay was successfully used to determine bradykinin concentrations in plasma samples. Intensive care unit patients with septic shock had low concentrations of plasma bradykinin during both shock and recovery phases.Peer reviewe

Comparison of serum serotonin and serum 5-HIAA LC-MS/MS assays in the diagnosis of serotonin producing neuroendocrine neoplasms : A pilot study

Background: Serotonin (5-hydroxytyramine) is a mediator of gastrointestinal smooth muscle contraction, and is secreted by neuroendocrine neoplasms (NENs). We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for serum serotonin to be used in NEN diagnostics and follow-up. Methods: We used serum samples from healthy volunteers (n = 31) and patients suspected or monitored for NEN (n = 98). Serotonin-D-4 internal standard was added to samples before solid phase extraction (SPE) and quantification by LC-MS/MS. The effects of sample handling and preparation on serotonin stability were studied. Finally, we established a provisional reference range for serum serotonin and compared our assay with serum 5hydroxyindoleacetic acid (5-HIAA) for detection of NENs. Results: Our assay is sensitive and has a wide linear range (10-10,000 nmo1/1). Serum serotonin is stable for 7 days at room temperature and for 3 months at -20 degrees C. Sampling temperature is not critical. Normal range for serum serotonin was 270-1490 nmo1/1. We found that serum serotonin and 5-HIAA performed equally well as diagnostic tests for NENs. Conclusions: Our LC-MS/MS assay for serum serotonin is well suited for clinical research and patient diagnostics. Our results confirm that it can complement 5-HIAA in diagnosis of NENs.Peer reviewe

Quantitative bile and serum proteomics for the screening and differential diagnosis of primary sclerosing cholangitis

BackgroundPrimary sclerosing cholangitis (PSC) is a chronic liver disease characterized by biliary strictures, cholestasis, and a markedly increased risk of cholangiocarcinoma. New markers for the screening and differential diagnosis of PSC are needed. In this pilot study, we have analyzed both the bile and serum proteomic profiles of 80 PSC patients and non-PSC controls (n = 6 for bile and n = 18 for serum).AimThe aim of this study was to discover candidates for new biomarkers for the differential diagnosis of PSC.MethodsBile and serum samples were processed and subsequently analyzed using ultra performance liquid chromatography-ultra definition mass spectrometry (UPLC-UDMSE). Further analysis included statistical analyses such as receiver operating characteristic curve analysis as well as pathway analysis using Ingenuity Pathway Analysis.Results and conclusionsIn bile, we discovered 64 proteins with significantly different levels between the groups, with fold changes of up to 129. In serum, we discovered 112 proteins with significantly different levels. Receiver operating characteristic curve analysis found multiple proteins with high area under the curve values, up to 0.942, indicating that these serum proteins are of value as new non-invasive classifiers of PSC. Pathway analysis revealed multiple canonical pathways that were enriched in the dataset, which have roles in bile homeostasis and metabolism. We present several serum proteins that could serve as new blood-based markers for the diagnosis of PSC after further validation. The measurement of serum levels of these proteins could be of use in the screening of patients with suspected PSC.Peer reviewe

Preoperative Radiotherapy Leads to Significant Differences in the Plasma Protein Profile of Rectal Cancer Patients

Introduction:Colorectal cancer (CRC) is the third most common cancer worldwide, accounting for 10% of the global cancer burden. Rectal cancer accounts for around 30% of CRC cases, and patients with resectable rectal cancer are often given preoperative radiotherapy (PRT) to reduce the rate of local recurrence. The human plasma proteome is an exceptionally complex proteome and ideal to study due to its ability to reflect the presence of diseases such as cancer and the ease of obtaining blood samples. Previous proteomic studies involving rectal cancer patients have mostly focused on the identification of proteins involved in resistance to radiotherapy.Objective:The aim of this study was to investigate the overall effects of PRT on plasma protein expression in rectal cancer patients, as there is a lack of such studies.Methods:Here, we have used mass spectrometry and subsequent statistical analyses to analyze the plasma samples of 30 rectal cancer patients according to PRT status (positive or negative) and tumor stage (II or III).Results and Conclusions:We discovered 42 proteins whose levels differed significantly between stage II and III rectal cancer patients who did or did not receive PRT. This study shows that PRT, although localized to the pelvis, leads to measurable, tumor stage-specific changes in plasma protein expression. Future studies of plasma proteins should, when relevant, take this into account and be aware of the widespread effects that PRT has on the plasma proteome.Peer reviewe

Plasma protein expression differs between colorectal cancer patients depending on primary tumor location

Colorectal cancer (CRC) includes tumors in the right colon, left colon, and rectum, although they differ significantly from each other in aspects such as prognosis and treatment. Few previous mass spectrometry-based studies have analyzed differences in protein expression depending on the tumor location. In this study, we have used mass spectrometry-based proteomics to analyze plasma samples from 83 CRC patients to study if differences in plasma protein expression can be seen depending on primary tumor location (right colon, left colon, or rectum). Differences were studied between the groups both regardless of and according to tumor stage (II or III). Large differences in plasma protein expression were seen, and we found that plasma samples from patients with rectal cancer separated from samples from patients with colon cancer when analyzed by principal component analysis and hierarchical clustering. Samples from patients with cancer in the right and left colon also tended to separate from each other. Pathway analysis discovered canonical pathways involved in lipid metabolism and inflammation to be enriched. This study will help to further define CRC as distinct entities depending on tumor location, as shown by the widespread differences in plasma protein profile and dysregulated pathways.Peer reviewe

Label-free plasma proteomics identifies haptoglobin-related protein as candidate marker of idiopathic pulmonary fibrosis and dysregulation of complement and oxidative pathways

Idiopathic pulmonary fibrosis (IPF) is a lung parenchymal disease of unknown cause usually occurring in older adults. It is a chronic and progressive condition with poor prognosis and diagnosis is largely clinical. Currently, there exist few biomarkers that can predict patient outcome or response to therapies. Together with lack of markers, the need for novel markers for the detection and monitoring of IPF, is paramount. We have performed label-free plasma proteomics of thirty six individuals, 17 of which had confirmed IPF. Proteomics data was analyzed by volcano plot, hierarchical clustering, Partial-least square discriminant analysis (PLS-DA) and Ingenuity pathway analysis. Univariate and multivariate statistical analysis overlap identified haptoglobin-related protein as a possible marker of IPF when compared to control samples (Area under the curve 0.851, ROC-analysis). LXR/RXR activation and complement activation pathways were enriched in t-test significant proteins and oxidative regulators, complement proteins and protease inhibitors were enriched in PLS-DA significant proteins. Our pilot study points towards aberrations in complement activation and oxidative damage in IPF patients and provides haptoglobin-related protein as a new candidate biomarker of IPF.Peer reviewe

Identification of several plasma proteins whose levels in colorectal cancer patients differ depending on outcome

Abstract Colorectal cancer (CRC) stands for 10% of the worldwide cancer burden and has recently become the second most common cause of cancer death. The 5-year survival rate depends mainly on stage at diagnosis. Mass spectrometric proteomic analysis is widely used to study the plasma proteome, which is complex and contains multitudes of proteins. In this study, we have used Ultra Performance Liquid Chromatography-Ultra Definition Mass Spectrometry (UPLC-UDMSE)-based proteomics to analyze plasma samples from 76 CRC patients. We identified several plasma proteins, such as CP, TVP23C, FETUB, and IGFBP3, of which altered levels led to significant differences in survival, as seen by Cox regression and Kaplan-Meier analysis. Additionally, during Cox regression analysis, samples were adjusted for age and/or tumor stage, enabling stringent analysis. These proteins, although in need of further validation, could be of use during patient follow-up, as their levels can non-invasively be measured from blood samples, and could be of use in predicting patient outcome. Several of these proteins additionally have roles in metabolism and inflammation, two processes central to the development and progression of cancer, further indicating their importance in cancer.Peer reviewe

Urinary extracellular vesicles carry multiple activators and regulators of coagulation

Cells shape their extracellular milieu by secreting intracellular products into the environment including extracellular vesicles which are lipid-bilayer limited membrane particles. These vesicles carry out a range of functions, including regulation of coagulation, via multiple contributor mechanisms. Urinary extracellular vesicles are secreted by various cells, lining the urinary space, including the nephron and bladder. They are known to have procoagulant properties, however, the details of this function, beyond tissue factor are not well known. The aim of the study was to access the role of urinary extracellular vesicles in impacting coagulation upon supplementation to plasma. This could indicate their physiological function upon kidney injury or pathology. Supplementation to standard human plasma and plasmas deficient in various coagulation factors was used for this purpose, and calibrated automated thrombogram (CAT (R)) was the major technique applied. We found that these vesicles contain multiple coagulation-related factors, and their lipid composition affects coagulation activities of plasma upon supplementation. Remarkably, these vesicles can restore thrombin generation in FVII, FVIII, FIX and FXI -deficient plasmas. This study explores the multiple roles of urinary extracellular vesicles in coagulation in in vitro blood coagulation and implies their importance in its regulation by several mechanisms.Peer reviewe