4 research outputs found
Ăn skreddersydd immunocapture-LC-MS/MS-metode for diagnose av smĂĽcellet lungekreft : Multipleksing og differensiering av ProGRP- og NSE-varianter
Smücellet lungekreft er den mest aggressive formen for lungekreft med en overlevelsesrate pü mindre enn 5 % etter 5 ür. Sykdommen responderer imidlertid godt pü behandling hvis initiert pü et tidlig stadium. Det er derfor viktig ü utvikle gode diagnostiske verktøy som tillater tidlig diagnose og behandlingsmonitorering. Sammen med røntgen og CT-scanning kan bruk av spesifikke og sensitive biomarkører for smücellet lungekreft bidra til dette.
Neuronspesifikk enolase (NSE) er den mest etablerte biomarkøren for smücellet lungekreft. Samtidig bestemmelse av progastrinfrigjørende peptid (ProGRP), som er en lovende biomarkør for differensiert diagnose av smücellet lungekreft, vil vÌre mer nøyaktig da ProGRP viser høyere sensitivitet og spesifisitet enn NSE. Begge proteinene bestemmes i dag separat ved bruk av immunometriske metoder. Disse metodene er imidlertid utsatt for kryssreaktivitet som kan resultere i falske positive og -negative resultater, i tillegg til at de ikke skiller mellom de ulike isoformene av proteinene.
Hensikten med denne oppgaven var ĂĽ fusjonere to tidligere utviklede validerte metoder for deteksjon av ProGRP og NSE til ĂŠn enkelt metode uten ĂĽ pĂĽvirke resultatene som metodene gir separat. Begge metodene involverer immunoaffinitetsekstraksjon og âbottom-upâ LC-MS/MS-analyse. En metode som tillater samtidig bestemmelse av ProGRP-isoformer og NSE-isoenzymer, vil vĂŚre en arbeidsbesparende metode som tillater differensiert bestemmelse av to komplementĂŚre biomarkører for smĂĽcellet lungekreft.
For ü evaluere den nye metoden ble det i denne oppgaven utført en grundig sammenligning av den fusjonerte metoden og de separate metodene. Forsøkene som vil bli presentert viser at de endrede forholdene ved fusjoneringen fortsatt gir god reproduserbarhet og utbytte. Forsøkene viser følgende effekter av endrede forhold; 1) det relative utbyttet av de respektive markørene ble ikke püvirket ved addisjon av ulike magnetiske kuler til varierende spikede løsninger, 2) reduksjon, varme og alkylering før tryptisk proteolyse ga økt signalintensitet for samtlige signaturpeptider sammenlignet med ingen pre-tryptisk behandling, 3) ProGRP- og NSE-konsentrasjoner ble sammenlignet ved varierende konsentrasjon av den andre markøren der en uparet t-test viste ingen signifikant forskjell i mült signalintensitet som tilsier at ProGRP- og NSE-konsentrasjoner kan bestemmes uavhengig av hverandres konsentrasjon, 4) kalibreringskurver for samtlige signaturpeptider ble harmonisert i serum hvilket tillater samtidig bestemmelse av ukjente ProGRP- og NSE-konsentrasjoner i pasientprøver.
Vellykket fusjon av de to metodene for ProGRP og NSE, inkludert deres isoformer og isoenzymer, viser at metoden gir pülitelig bestemmelse og differensiering av klinisk relevante nivüer av to etablerte, komplementÌre biomarkører for smücellet lungekreft. Ved analyse av pasientprøver med den fusjonerte metoden ble resultatene funnet sammenfallende med eksisterende immunometriske assays og dette bekrefter at metoden kan vÌre et egnet diagnoseverktøy for smücellet lungekreft
Matrix-Assisted Ionization and Tandem Mass Spectrometry Capabilities in Protein Biomarker Characterization - An Initial Study Using the Small Cell Lung Cancer Biomarker Progastrin Releasing Peptide as a Model Compound
This initial study evaluates vacuum matrix-assisted ionization (vMAI) mass spectrometry (MS) for identification and determination of tryptic peptides from the biomarker protein progastrin releasing peptide (ProGRP). Similar peptides and charge states were observed as in liquid chromatography (LC) electrospray ionization (ESI) MS. The prolonged ion duration in vMAI with similar charge states as in ESI was advantageous for determining the MS/MS fragmentation conditions compared to MAI. It is assumed that the vacuum ionization conditions lower the detection limits of the experiment. This may be the reason vMAI combined with high resolution MS enabled detection of tryptic peptides from more digested proteins than MAI selected reaction monitoring MS. Additionally, MAI ion mobility spectrometry MS (MAI-IMSâMS) was evaluated for differentiation of intact protein isoforms, successfully enabling differentiation of the isoforms by drift time selection. Examples are both shown for model proteins bovine serum albumin, cytochrome C, and lysozyme and the clinically relevant small cell lung cancer protein biomarker ProGRP, which exists in three isoforms. Coupling with the vacuum ionization conditions using a dedicated vacuum-probe source MAI enables information to be extracted readily as with conventional approaches, just faster
To elute or not to elute in immunocapture bottom-up LCâMS
Immunocapture-based bottom-up LCâMS is a promising technique for the quantification of low abundant proteins. Magnetic immunocapture beads provide efficient enrichment from complex samples through the highly specific interaction between the target protein and its antibody. In this article, we have performed the first thorough comparison between digestion of proteins while bound to antibody coated beads versus after elution from the beads. Two previously validated immunocapture based MS methods for the quantification of pro-gastrin releasing peptide (ProGRP) and human chorionic gonadotropin (hCG) were used as model systems. The tryptic peptide generation was shown to be protein dependent and influenced by protein folding and accessibility towards trypsin both on-beads and in the eluate. The elution of proteins bound to the beads was also shown to be incomplete. In addition, the on-beads digestion suffered from non-specific binding of the trypsin generated peptides. A combination of on-beads digestion and elution may be applied to improve both the quantitative (peak area of the signature peptides) and qualitative yield (number of missed cleavages, total number of identified peptides, coverage, signal intensity and number of zero missed cleavage peptides) of the target proteins. The quantitative yield of signature peptides was shown to be reproducible in all procedures tested
Immunocapture sample clean-up in determination of low abundant protein biomarkers-a feasibility study of peptide capture by anti-protein antibodies
Immunocapture in mass spectrometry based targeted protein analysis using a bottom-up workflow is nowadays mainly performed by target protein extraction using anti-protein antibodies followed by tryptic digestion. Already available monoclonal antibodies (mAbs) which were developed against intact target proteins (anti-protein antibodies) can capture proteotypic epitope containing peptides after tryptic digestion of the sample. In the present paper considerations when developing a method for targeted protein quantitation through capture of epitope containing peptides are discussed and a method applying peptide capture by anti-protein antibodies is compared with conventional immunocapture MS. The model protein used for this purpose was progastrin releasing peptide (ProGRP), a validated low abundant biomarker for Small Cell Lung Cancer with reference values in serum in the pg mLâ1 range. A set of mAbs which bind linear epitopes of ProGRP are available, and after a theoretical consideration, three mAbs (E146, E149 and M18) were evaluated for extraction of proteotypic epitope peptides from a complex sample. M18 was the best performing mAb for peptide capture by anti-protein antibodies, matching the LOD (54 pg mLâ1) and LOQ (181 pg mLâ1) of the existing conventional immunocapture LC-MS/MS method for determination of ProGRP. Peptide and protein capture using the same mAb were also compared with respect to sample clean-up, and the peptide capture workflow yielded cleaner extracts and therewith less complex chromatograms. Analysis of five patient samples demonstrated that peptide capture by anti-protein antibodies can be used for the determination of various levels of endogenously present ProGRP