Presentation on theme: "Micromass Quattro Ultima triple quadrupole mass spectrometric detector HPLC system (LC) Electrospray ionisation source (-ve & +ve ion) Photodiode array."— Presentation transcript:
Micromass Quattro Ultima triple quadrupole mass spectrometric detector HPLC system (LC) Electrospray ionisation source (-ve & +ve ion) Photodiode array detector (PDA) Fluorescence detector The Equipment LC-MS/MS
A little bit about triple quadrupole mass spectrometry.
A little bit about electrospray ionisation.
Measurement of protein mass by triple quadrupole MS. Protein (pure) sample (prepared by investigator) ■Multiple-charged ion series - deconvolution gives molecular masses ■Intractable analysis for complex protein mixtures. Limited mass resolution. Mass spectrometer Sample infusion Infusion pump Electrospray source Parent ions Protein n+ Protein (n+x)+ Mass analyser-2 OFF Photomultiplier detector Response Mass analyser-1 Collision cell
Applications: Peptide mapping of haemoglobin modified by methylglyoxal
Detection of protein biomarkers by LC-MS/MS: Multiple reaction monitoring (MRM) HPLC Mass analyser-1 Parent ion Electrospray source Biomarker + Mass analyser-2 Biomarker fragment Collision cell Fragment ion + ■High specificity ■(LC, MS1 and MS2 resolution) ■High sensitivity ■Biomolecule compatible Mass spectrometer Enzymatic hydrolysate (prepared by investigator) Photomultiplier detector Response ■ Biomarker screening in 75 min per sample.
Advanced glycation endproducts LC-MS/MS with stable isotope-substituted internal standards
Detection of protein biomarkers by LC-MS/MS: Calibration, sample de-lipidification, ultrafiltration & enzymatic hydrolysis Delipidification and AGE fractionation ■Ultrafiltration to separate protein AGE residues and free AGEs ■Ether or methanol/chloroform extraction Analytical performance ■Limits of detection: 20 – 500 fmol. ■Recoveries: >80%; % for amino acids ■Interbatch c.v.: <10% (n = 6) Enzymatic digestion: ■Pepsin (+ thymol) ■Pronase E (under nitrogen, penicillin and streptomycin added) ■Prolidase and aminopeptidase (under nitrogen) Internal standardisation and calibration ■Standards and stable isotope-substituted standards e.g. CML and [ 13 C 6 ]CML, MG-H1 and [ 15 N 2 ]MG-H1
Detection of protein biomarkers by LC-MS/MS: Retention of amino acids and AGEs and use of column switching Hypercarb graphitic columns retain underivatised amino acids, allowing for diversion of non-volatile salts to waste. Non-volatile salts to waste Hypercarb column (2.1 x 250 mm) Hypercarb column (2.1 x 50 mm) Sample To MS/MS Column switching facilitates elution of hydrophobic analytes and column washing. Switching valve
Examples of detection by multiple reaction monitoring (MRM): CML CML detected in plasma protein of a normal healthy human control subject.
Examples of detection multiple reaction monitoring (MRM): Methylglyoxal-derived hydroimidazolone MG-H1 detected in rat retinal protein hydrolysate of a STZ diabetic rat.
Arg H 2 CO 2, NH 2 C(=NH)NH 2 Lys H 2 CO 2, NH 3 Met H 2 CO 2 MetSO CH 3 SOH CML NH 2 CH 2 CO 2 H MG-H NH 2 CH(CO 2 H)CH 2 CH=CH 2 Pent NH 2 CH(CO 2 H)CH 2 CH 2 CH=CH 2 Analyte Rt Parent Ion Fragment ion CE Natural Fragment loss (min) (Da) (Da) (eV) Mass spectrometric multiple reaction monitoring detection of protein biomarkers
Peptide mapping to identify sites of protein modification Mass spectrometer Mass analyser-1 Collision cell Electrospray source Parent ions Peptides + Tryptic digest of protein sample (prepared by investigator) HPLCPDA Resolution of peptide fragments by LC Mass analyser-2 OFF Photomultiplier detector Single ion response for each peptide Peptide map Biolynx match of peptide M+ with theoretical digest. Locate modified peptide M+ ion Peptide mapping to identify glycation sites.
Glycation of human serum albumin by methylglyoxal Location of glycation sites by LC-MS peptide mapping MS detection of peptide fragments by LC-MS and quantitation of the MS response Peptides are partially resolved by HPLC with ODS chromatography and detected by positive ion electrospray MS. Limited proteolysis of MG min -HSA and HSA control Reduction of disulphide bonds with dithiothreitol. S-Alkylation of cysteine thiols by iodoacetamide. Digestion with trypsin (and independently with Glu-C for corroboration).
MS detection of peptide fragments by LC-MS and quantitation of the MS response Peptide responses are normalised to the C-terminal peptide (LVAASQAALGL). Loss of peptides in MG min -HSA digest was quantified by the mean normalised peptide response for MG min -HSA, relative to HSA control (mean c.v. = 11%). This is assumed due to glycation The glycated peptides were also detected as modified dipeptides (resistant to proteolysis in tryptic maps). Modification of arg-410 Peptide(T52) FQNALLVR
LC-MS/MS peptide mapping can also be used to locate glycation, oxidation and nitration markers Location of MG-H1 residues in human serum albumin modified minimally by methylglyoxal Ion chromatograms for peptide T52 (containing R410) Ion chromatograms for dipeptide T52-53 (containing MG-H1-410) Predicted mass of T52-53 FQNALLVRMG-H1YTK ; found peptide mass Da.
Glycation of human serum albumin by methylglyoxal Location of glycation sites by LC-MS peptide mapping R410 R218 R114 R186 R428 ArgMG-H1 (mol%) Modification hotspot: Arg-410 Drug binding site 2. Active site of esterase activity.