Peptide hormones: the sensitivity quest and sensitivity questions. Eduard Rogatsky Albert Einstein College of Medicine Einstein-Montefiore Institute for Clinical & Translational Research Bronx, NY

Pro-insulin is cleaved into Insulin and C-peptide

Insulin but not C-peptide is cleared by the liver INSULIN GLUCOSE C-peptide Pancreas Liver Venous Blood Portal Vein Pro-insulin is synthesized in the pancreatic beta cells Pro-insulin cleaved to insulin and C-peptide. Insulin and C-peptide are secreted in a 1:1 molar ratio. Measurement of C-peptide used to calculate insulin secretion

Insulin itself should not be used for quantitation in attempt to understand its biosynthesis rates due to high insulin clearance Pro-insulin C-peptide is the best target to asses the insulin biosynthesis rates

Obese Fasting:1-2 ng/ml; Stimulated: ng/ml F: 5-10 µ u/ml; S: µ u/ml Normal Fasting:1-2 ng/ml; Stimulated: ng/ml F: 2-5 µ u/ml; S: µ u/ml Type 2 Diabetes Fasting:1-2 ng/ml; Stimulated: 2 -3 ng/ml F: 5-10 µ u/ml; S: µ u/ml Type 1 DiabetesLess 0.5 ng/ml Less 2 µ u/ml C-peptide and Insulin levels 1 µu/ml = 6 pmol/L 10 µu/ml = 350 pg/ml C-pept ide Insulin

LC/MS analysis of low level analytes in complex matrices The most common ways of improving sensitivity: Use more sample Use capillary/nano chromatography Purchase a new generation instrument Improve analyte purity

1D vs 2D chromatography

2D RP-RP Method: Valve Position 1 Binary Pump 1 Binary Pump Autosampler Plug LC/MS column 2 Column 1 Mixing Tee Column compartment G1316A (Agilent) Line A Closed Waste Mass Spectrometer Valve 2 Jupiter 5u 100x2mm C5 300A (Phenomenex) Polaris 5u 50x2mm C18 A 180A (Varian ) 0.1% TFA

2D RP-RP Method: Valve Position 2 Binary Pump 1 Waste Binary Pump Autosampler Plug Mass Spectrometer LC/MS column 2 Column 1 Valve 2 Mixing Tee Column compartment G1316A (Agilent) Line A Open Jupiter 5u 100x2mm C5 300A (Phenomenex) 0.1% TFA Polaris 5u 50x2mm C18 A 180A (Varian)

Column Switching Window Overlapped gradients - profile Anti- carryover flush Eduard Rogatsky and Daniel Stein. Two dimensional reverse phase - reverse phase chromatography: a simple and robust platform for sensitive peptide quantitative analysis by LC/MS. Hardware design. Journal of Separation Science. 2006, 29,

Time, min 0.0 M+0 M+14 M+7 M+30 Highly rugged, accurate and reproducible assay ~70 injections of plasma samples, injections of urine per study. LLOQ better than 50 pg/ml (5 pg on column) C-peptide species Requirements for LC/MS assay of C-peptide isotopomers

125 ms Area ratio of M+7/14. 2D/MRM %RSD = 13.3; 2D SIM%RSD = 3.3; A, M+14; MRMB, M+14; MRM C, M+14; MRM D, M+14; SIM S/N 20-25

Linearity of isotope dilution assay of urinary C-peptide by 1D SIM and 1D MRM

Linearity of quantitation of stable isotope labeled IS by 2D SIM in background of 20% urine

S/N =14 Quantitative analysis by 2D/SIM of 5 pg on column (1.6 fmole; 167pg/ml) of m+30 stable isotope labeled internal standard on the background of a 100 fold excess of unlabeled urinary C-peptide (20% urine)

IS C-pep Type 1 Diabetes, ~ 15 pg/ml of C-peptide

Analysis of 150 pg on column of m+30 stable isotope labeled internal standard in background of 40% urine 2D chromatography and SIM. S/N = 264.6; peak area 4.75e+05 2D SIM outperform 1D MRM 1D chromatography and MRM. S/N = 117.1; peak area 2.55e+03

2D SIM vs 2D MRM, SPE plasma QC, SIM, S/N 963; p/p 123 Same samp, MRM, S/N 370; p/p 61

Effect of Multiple Stages of Mass Spectrometry on Signal-to-Noise Signal Noise S/N Stages of Mass Spectrometry The figure is adapted from F.W. McLafferty’s book “Tandem Mass Spectrometry” published in 1983

CID of peptides: multiple fragments formation Product scan of ; CE 35ev Y1, ~ 1.5% yield y2 y3 y4 y5 y6 y9 b7 b6 y10 b1 b2

Impact of dwell time: C-peptide 50 ms SIM 100 ms SIM Area ratio of M+7/14. 2D/SIM 100ms %RSD = 6.7; 50 ms %RSD = 6.4;

Impact of dwell time - MRM 50 ms MRM; 19% RSD 100 ms MRM; 12% RSD peak definitions problems

Impact of dwell time - MRM 25 ms MRM; 22% RSD peak definitions troubles

Impact of dwell time: SIM vs MRM 10 ms MRM; 53% RSD 10 ms SIM; 11% RSD

27 Fused-Core Ascentis Express Particles Comparison of architecture of Fused- Core™ particles vs. porous 3 µm particles

2x50 Polaris C18 3µm 180 A versus 2x30 Ascentis Express Peptide 160A S/N 170 S/N 40 Low loading capacity –the main limitation factor of the Fused-Core material – was overcome by using 2D chromatography. Only small, C-Peptide containing fraction was loaded [transferred] onto the column.

Advantages of PEEKsil Fused Silica Lined PEEK tubing Smooth Wall Surface Precision of Inside Diameter Inertness Pre-cut

100 µm ID PEEKsil vs. 100 µm PEEK

100 µm ID PEEKsil vs. 125 µm PEEK 20% S/N gain 4.05µl vs 6.35µl

Conclusions We document for the first time that insulin biosynthesis rates can be monitored in vivo We demonstrate applicability of fused –core column in analysis of biological samples (diluted urine and SPE –pre-purified plasma or serum) using 2 dimensional chromatography. Sample load onto the first dimension [5 µm] allows large sample volume to be injected Use of PEEKSIL tubing improves S/N

Funding: NIH: MO1-RR12248 NIH: R01 DK Technical Support: Agilent Technologies Applied Biosystems Phenomenex Supelco SGE Acknowledgements