One column method SPE AcN/Phospate sample

One column method SPE AcN/Phospate sample

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 C-PEPTIDE G LY G LU LEU ALA LEU PRO G LN LEU SER G LY ALA G LY PRO G LY G LY G LY LEU G LU VAL G LN G LY VAL G LN LEU ASP G LU SER ALA 50 60 GLY LEU 40 GLU LEU ALA LEU PRO GLN LEU SER GLY ALA GLY PRO GLY GLY GLY LEU GLU VAL GLN GLY VAL GLN LEU G LU = Leucine G LN ASP SER GLU LEU ALA GLN GLU LYS ARG GLY

S--S 70 A-Chain ARG 80 ILE VAL GLU GLN CYS CYS THR SER ILE CYS SER LEU TYR GLN LEU GLU ASN TYR CYS ASN ARG - COO- THR LYS s s Protease catalyzed hydrolysis A-Cha in S- - S PRO s s 30 I LE THR B-Chain VAL G LU G LN CYS CYS THR SER I LE CYS SER LEU TYR G LN LEU G LU ASN TYR CYS ASN - COO- TYR VAL ASN GLN HIS LEU CYS GLY SER HIS LEU VAL GLU ALA LEU TYR LEU VAL CYS GLY GLU ARG GLY PHE 10 PHE 1

s s 20 s THR s PROINSULIN LYS PRO B-Chain VAL ASN G LN HI S LEU CYS G LY SER HI S LEU THR VAL G LU ALA LEU INSULIN TYR LEU VAL CYS G LY G LU ARG G LY PHE PHE TYR Insulin but not C-peptide is cleared by the liver 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 Venous Blood Liver Portal Vein INSULIN C-peptide GLUCOSE Pancreas 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 C-peptide and Insulin levels

C-peptide Insulin Obese Fasting:1-2 ng/ml; Stimulated: 4 -15 ng/ml F: 5-10 u/ml; S: 50-200 u/ml Normal Fasting:1-2 ng/ml; Stimulated: 2 - 5 ng/ml F: 2-5 u/ml; S: 10-30 u/ml Type 2 Diabetes Fasting:1-2 ng/ml; Stimulated: 2 -3 ng/ml F: 5-10 u/ml; S: 20-100 u/ml Type 1 Diabetes Less 0.5 ng/ml Less 2 u/ml 1 u/ml = 6 pmol/L 10 u/ml = 350 pg/ml 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 +ESI MRM Frag=300.0V [email protected] (1007.6 -> 147.1) qc 1d 10 ul006.d Noise (RMS) = 1.30; SNR (2.229min) = 122.5 x10 2 6.2 1 12 2 6 5.8 5.6 5.4 5.2 5 4.8 4.6 4.4 4.2 4 3.8 3.6 3.4 3.2 3 2.8 2.6 2.4 2.2 321159 2 1.8 1.6 1.4 1.2 1 0.8 0.6 52 0.4 0.2 0 0.1

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 Counts vs. Acquisition Time (min) 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 2D RP-RP Method: Valve Position 1 Binary Pump 1 Column compartment G1316A (Agilent) Jupiter 5u 100x2mm C5 300A (Phenomenex) Autosampler 0.1% TFA Column 1 Mixing Tee Binary Pump 2 Line A Closed 1 6

2 5 3 4 Plug LC/MS column 2 Polaris 5u 50x2mm C18 A 180A (Varian ) Valve 2 Waste Mass Spectrometer 2D RP-RP Method: Valve Position 2 Column compartment G1316A (Agilent) Binary Pump 1 Jupiter 5u 100x2mm C5 300A (Phenomenex) Autosampler 0.1% TFA Column 1 Mixing Tee Binary Pump 2 1 Line A Open 6 2 5 3 4 Plug LC/MS column 2 Polaris 5u 50x2mm C18 A 180A (Varian) Valve 2 Waste Mass Spectrometer Overlapped gradients - profile 100 %B 90 80 Pump 1 Column Switching Window 70 60 50 40 30 Pump 2 20 10 0 0 5 10 Time, min

Anticarryover flush 15 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, 538-546. Requirements for LC/MS assay of C-peptide isotopomers C-peptide species Highly rugged, accurate and reproducible assay ~70 injections of plasma samples, 60 -80 injections of urine per study. M+0 M+30 M+7 M+14 0.0 Time, min LLOQ better than 50 pg/ml (5 pg on column) XIC of +MRM (4 pairs): 1012.4/147.1 amu from Sample 30 (QC_MRM) of 08112008SET1.wiff (Turbo... XIC of +MRM (4 pairs): 1012.4/147.1 amu from Sample 31 (QC_MRM) of 08112008SET1.wiff (Turbo... Max. 88.0 cps. 12.02 55 -0.03 88 85 Max. 56.0 cps. 0.05 50 80 A, M+14; MRM 75 70 B, M+14; MRM 45 40 65 60 50 45 12.05 40 35 I n t e n s it y , c p s In t e n s i ty , c p s 55 30 25 35 20 ! - Noise - ! 30 -0.10

25 15 20 ! - Noise - ! 10 15 10 -0.27 0.23 5 5 0 11.3 11.4 11.5 11.6 11.7 11.8 11.9 12.0 12.1 12.2 Time, min 12.3 12.4 12.5 12.6 12.7 12.8 0 12.9 XIC of +MRM (4 pairs): 1012.4/147.1 amu from Sample 27 (QC_MRM) of 08112008SET1.wiff (Turbo... 75 60 11.5 11.6 11.7 11.8 11.9 12.0 12.1 12.2 Time, min 12.3 12.4 12.5 12.6 12.7

12.8 12.9 Max. 7656.0 cps. 7500 S/N 20-25 7000 12.03 70 65 11.4 11.23 0.03 80 11.3 XIC of +Q1 MI (4 ions): 1012.4 amu from Sample 8 (QC_SIM) of 08112008SET1.wiff (Turbo Spra... Max. 80.0 cps. 6500 12.07 6000 C, M+14; MRM D, M+14; SIM 5500 55 5000 In te n s ity , c p s I n te n s ity , c p s 50 45 40 35 30 11.54 4500 4000 11.98 11.71 3500 12.37 12.48 12.63 3000 12.77 2500 25 12.87 2000 20 0.10 15 ! - Noise - !

1500 0.90 1000 10 500 5 0 0 11.3 11.4 11.5 11.6 11.7 11.8 11.9 12.0 12.1 12.2 Time, min 12.3 12.4 12.5 12.6 12.7 12.8 12.9 11.3 11.4 11.5 11.6 11.7 11.8 11.9 12.0 12.1 12.2 Time, min 12.3 12.4 12.5 12.6 12.7 12.8 12.9 13.0 125 ms Area ratio of M+7/14. 2D/MRM %RSD = 13.3; 2D SIM%RSD = 3.3; Linearity of isotope dilution assay of urinary C-peptide by 1D SIM and 1D MRM C-peptide/IS area ratio 9.00 8.00 1D SIM 7.00 1D MRM

6.00 Linear (1D MRM) 5.00 4.00 y = 0.2012x R2 = 0.9969 3.00 2.00 1.00 0.00 0 10 20 30 40 urine concentration (%, w /w ) 50 Linearity of quantitation of stable isotope labeled IS by 2D SIM in background of 20% urine 3.00E+06 IS area, cps 2.50E+06 y = 4063x R2 = 0.9995 2.00E+06 1.50E+06 1.00E+06 5.00E+05 0.00E+00 0 100 200 300 400 IS, pg on column 500 600 700 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) XIC of +Q1 MI (2 ions): 1007.6 amu from Sample 11 (5pg IS on column; 2D Q1+) of 09242005S... Max. 2.6e5 cps. 13.96 2.6e5 2.4e5 1 1- Human C-peptide 2.2e5 2.0e5 1.8e5 S/N =14 In te n s ity , c p s 1.6e5 1.4e5 1.2e5 1.0e5

8.0e4 6.0e4 4.0e4 2.0e4 0.0 2 - isotope labeled internal standard 12.6 12.8 13.0 13.2 13.4 13.6 13.8 Time, min 2 14.0 14.2 14.4 14.6 14.8 Type 1 Diabetes, ~ 15 pg/ml of C-peptide XIC of +Q1 MI (4 ions): 1007.7 amu from Sample 18 (Diab Samp 4 500-1500 b) of 11082011SET1.wiff (Turbo Spray) Max. 1.0e4 cps. 2.8e4 2.6e4 IS 2.4e4 2.2e4 2.0e4 1.8e4 In te n s ity , c p s 1.6e4 1.4e4 1.2e4 1.0e4 6.01 10.67 8000.0 10.65 6000.0 11.89 4000.0 10.18 10.14 8.59 8.69 8.90 2000.0 0.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 10.85 12.30 9.20 9.479.63 9.5 Time, min

10.0 10.5 11.0 C-pep 11.93 11.27 11.5 12.0 12.5 12.96 2D SIM outperform 1D MRM Analysis of 150 pg on column of m+30 stable isotope labeled internal standard in background of 40% urine XIC of +MRM (2 pairs): 1017.6/147.2 amu from Sample 64 (1D MRM IS 50 mkl; Ur 400 mkl) of ... XIC of +Q1 MI (2 ions): 1017.6 amu from Sample 65 (400 mkl Ur, 50mkl IS; 2DQ1+) of 0924200... Max. 6.0e4 cps. 6.73 338 14.05 6.0e4 320 S/N = 264.6 S/N = 117.1 300 5.5e4 280 Peak Int.(Subt.)=5.7e+4 5.0e4 Max. 337.5 cps. Peak Int.(Subt.)=3.4e+2 260 1xStd.Dev.(Noise)=2.2e+2 4.5e4 240 1xStd.Dev.(Noise)=2.9e+0 220 4.0e4 Intensity, cps Intensity, cps 3.5e4 3.0e4 200 180 160 140 2.5e4 120 2.0e4 100 1.5e4 0.71

1.0e4 5000.0 -1.54 0.0 -3.47 80 -1.38 -1.16 -1.09 12.6 12.8 ! 13.0 -0.84 -0.77 13.31 - Noise - 13.2 13.4 40 0.39 -0.43 ! -0.28 13.6 13.8 Time, min 60 20 14.0 14.2 14.4 14.6 14.8 2D chromatography and SIM. S/N = 264.6; peak area 4.75e+05 0 -2.77 -2.52 4.38 -2.05 -1.40 -3.69 2.5 3.0 ! 3.5 4.0 - Noise - 4.5 5.0 5.5 0.30 -0.86 -0.38 ! 6.0 6.5 Time, min 7.0 1.03 1.16 1.51 1.66 2.32 7.5

9.0 8.0 8.5 9.5 1D chromatography and MRM. S/N = 117.1; peak area 2.55e+03 2D SIM vs 2D MRM, SPE plasma XIC of +Q1 MI (4 ions): 1007.7 amu from Sample 8 (QC_SIM) of 08112008SET1.wiff (Turbo Spray... Max. 1.3e5 cps. XIC of +MRM (4 pairs): 1007.7/147.1 amu from Sample 31 (QC_MRM) of 08112008SET1.wiff (Tur... 12.10 1900 1.3e5 Max. 1928.0 cps. 12.05 1800 1.2e5 1700 1.1e5 1600 1500 1.0e5 1400 9.0e4 1300 Intensity, cps Intensity, cps 8.0e4 7.0e4 1200 1100 1000 900 6.0e4 800 5.0e4 700 600 4.0e4 500 3.0e4 400 2.0e4 300 200 1.0e4 100 0.0 11.3 11.4 11.5 11.6 11.7 11.8 11.9 12.0 12.1 12.2 12.3 12.4 12.5 12.6 12.7 12.8 12.9 13.0 Time, min QC, SIM, S/N 963; p/p 123 0

11.3 11.4 11.5 11.6 11.7 11.8 11.9 12.0 12.1 12.2 12.3 12.4 12.5 12.6 12.7 12.8 12.9 Time, min 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. McLaffertys book Tandem Mass Spectrometry published in 1983 CID of peptides: multiple fragments formation +MS2 (1007.80): 299 MCA scans from Sample 1 (TuneSampleID) of MT20081029143110.wiff (Tur... 147.3 3.8e6 3.6e6 Max. 3.8e6 cps. Y1, ~ 1.5% yield 3.4e6 3.2e6 3.0e6 2.8e6 2.6e6 Product scan of 1007.8; CE 35ev 2.4e6 I n te n s it y , c p s 2.2e6 2.0e6 1.8e6 y2 1.6e6 1.4e6 260.5 y4 1.2e6 8.0e5 6.0e5 4.0e5 y9 404.5 1.0e6 b1 y3 130.2 2.0e5 100 y5 b2 282.4 347.2 386.3 414.4 534.6 558.3 183.0 201.0211.5 300.6 314.5 200 300 b6 y6 686.6 533.6

400 500 646.6 600 m/z, amu 668.5 700 b7 927.9 y10 785.8 717.7 767.8 800 855.0 909.6 900 970.8 1056.2 995.8 1008.3 1000 1100 Impact of dwell time: C-peptide Sample Name: "100 MS SIM" Sample ID: "" File: "03032009SET1.wiff" Peak Name: "1012.4 M+14" Mass(es): "1012.4 amu" Comment: "" Annotation: "" Sample Index: 5 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.000 ng/mL 1.4e4 Acq. Date: 3/3/2009 Acq. Time: 4:07:25 PM Modified: Proc. Algorithm: Bunching Factor: Noise Threshold: Area Threshold: Num. Smooths: Sep. Width: Sep. Height: Exp. Peak Ratio: Exp. Adj. Ratio: Exp. Val. Ratio: RT Window: Expected RT: Use Relative RT: Yes Analyst Classic 2 56.13 cps 280.65 cps 3 0.20 0.01 5.00 4.00 3.00 30.000 sec 11.591 min No Int. Type: Manual Retention Time: 11.571 Area: 1.649e+004 Height: 3.167e+003 Start Time: 11.469 End Time:

11.672 Sample Name: "50 ms SIM" Sample ID: "" File: "03032009SET1.wiff" Peak Name: "1012.4 M+14" Mass(es): "1012.4 amu" Comment: "" Annotation: "" Sample Index: 15 Sample Type: Unknown 1.3e4 Concentration: N/A Calculated Conc: 0.000 ng/mL Acq. Date: 3/3/2009 Acq. Time: 6:30:05 PM 1.3e4 1.3e4 Modified: Proc. Algorithm: Bunching Factor: Noise Threshold: Area Threshold: Num. Smooths: Sep. Width: Sep. Height: Exp. Peak Ratio: Exp. Adj. Ratio: Exp. Val. Ratio: RT Window: Expected RT: Use Relative RT: 1.3e4 1.2e4 1.2e4 1.1e4 min counts cps min min Yes Analyst Classic 2 56.13 cps 280.65 cps 3 0.20 0.01 5.00 4.00 3.00 30.000 sec 11.591 min No Int. Type: Manual Retention Time: 11.573 Area: 1.658e+004 Height: 3.533e+003 Start Time: 11.450 End Time: 11.722 1.1e4 1.0e4 1.2e4 1.2e4 1.1e4 1.1e4 min

counts cps min min 1.0e4 9500.0 9500.0 9000.0 9000.0 8500.0 8500.0 8000.0 8000.0 7500.0 7500.0 Intensity, cps Intensity, cps 7000.0 6500.0 7000.0 6500.0 11.57 6000.0 6000.0 11.57 5500.0 5500.0 5000.0 11.61 5000.0 4500.0 4500.0 4000.0 9.98 4000.0 3500.0 3500.0 9.97 3000.0 9.56 2500.0 9.39 2000.0 8.37 7.81 1500.0 1000.0 6.60 6.64 6.75 6.80 6.96 7.07 7.28 7.347.43

7.64 7.75 7.96 8.05 8.42 8.47 8.78 8.82 8.72 9.01 9.12 9.51 10.17 9.63 10.78 10.37 10.56 9.82 12.05 11.94 10.83 12.27 12.58 12.65 12.81 11.34 3000.0 9.43 2500.0 11.18 10.99 8.34 2000.0 9.30 8.41 8.72 8.79 8.85 9.02 10.18 10.25 9.62 9.71 9.93 11.00 10.42 10.76 11.68 11.35 11.20 11.94 12.29 12.64 12.53 11.27 9.18 9.34 1500.0 8.18 1000.0 500.0

0.0 12.07 10.79 12.09 500.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 Time, min 10.5 11.0 11.5 12.0 12.5 0.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 Time, min 100 ms SIM 50 ms SIM Area ratio of M+7/14. 2D/SIM 100ms %RSD = 6.7; 50 ms %RSD = 6.4; 12.0 12.5 12.82 Impact of dwell time - MRM Sample Name: "100 ms MRM" Sample ID: "" File: "03032009SET1.wiff" Peak Name: "1012.3 / 147.2" Mass(es): "1012.4/147.1 amu" Comment: "" Annotation: "" Sample Index: 37 Sample Unknown 11.54 Type: Concentration: N/A Calculated Conc: 0.000 ng/mL Acq. Date: 3/3/2009 58 Acq. Time:

11:43:56 PM Sample Name: "50 ms MRM" Sample ID: "" File: "03032009SET1.wiff" Peak Name: "1012.3 / 147.2" Mass(es): "1012.4/147.1 amu" Comment: "" Annotation: "" Sample Index: 49 Sample Type: Unknown Concentration: N/A 49 Calculated Conc: 0.000 ng/mL Acq. Date: 3/4/2009 48 Acq. Time: 9:52:42 AM 47 Modified: Yes Proc. Algorithm: Analyst Classic 46 Bunching Factor: 2 45 Noise Threshold: 1.45 cps Area Threshold: 7.27 cps 44 Num. Smooths: 3 Sep. Width: 0.20 43 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 42 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 41 RT Window: 80.000 sec 40 Expected RT: 11.591 min Use Relative RT: No 39 Int. Type: Manual 38 Retention Time: 11.543 min Area: 1.943e+002 counts 37 Height: 4.944e+001 cps Start Time: 11.421 min 36 End Time: 11.630 min 35 11.61 Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 1.45 cps Area Threshold: 7.27 cps Num. Smooths: 3 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio:

4.00 Exp. Val. Ratio: 3.00 RT Window: 80.000 sec Expected RT: 11.591 min Use Relative RT: No Int. Type: Manual Retention Time: 11.559 Area: 2.780e+002 Height: 5.950e+001 Start Time: 11.490 End Time: 11.679 11.56 56 54 52 50 48 46 min counts cps min min 44 42 34 33 40 32 38 31 30 36 29 peak definitions problems 28 27 32 Intensity, cps Intensity, cps 26 11.62 34 25 24 23 30 28 11.49 22 21 26 12.30 20

24 19 18 22 17 20 16 15 18 14 13 11.26 16 12 11 14 11.52 10 12 9 8 7 6 10 6.50 7.05 10.74 11.44 11.66 12.22 11.35 12.66 8 12.26 5 3 4 2 1 0 9.18 6 4 6.64 11.15 7.94 12.56 11.25 11.73 12.22 2

6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 Time, min 12.5 0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 Time, min 50 ms MRM; 19% RSD 100 ms MRM; 12% RSD Impact of dwell time - MRM Sample Name: "25 ms MRM" Sample ID: "" File: "03032009SET1.wiff" Peak Name: "1012.3 / 147.2" Mass(es): "1012.4/147.1 amu" Comment: "" Annotation: "" Sample Index: 54 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.000 ng/mL 115 Acq. Date: 3/4/2009 Acq. Time: 11:04:02 AM Sample Name: "25 ms MRM" Sample ID: "" File: "03032009SET1.wiff" Peak Name: "1012.3 / 147.2" Mass(es): "1012.4/147.1 amu" Comment: "" Annotation: "" Sample Index: 54 Sample Type: 11.55Unknown Concentration: N/A Calculated Conc: 0.000

ng/mL 115 Acq. Date: 3/4/2009 Acq. Time: 11:04:02 AM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 1.45 cps Area Threshold: 7.27 cps Num. Smooths: 3 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 80.000 sec Expected RT: 11.591 min Use Relative RT: No Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 1.45 cps Area Threshold: 7.27 cps Num. Smooths: 3 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 80.000 sec 11.56 Expected RT: 11.591 min Use Relative RT: No Int. Type: Manual Retention Time: 11.549 Area: 2.287e+002 Height: 1.184e+002 Start Time: 11.483 End Time: 11.633 110 105 100 95

min counts cps min min Int. Type: Manual Retention Time: 11.549 Area: 2.287e+002 Height: 1.184e+002 Start Time: 11.483 End Time: 11.633 90 85 110 105 100 95 90 min counts cps min min 85 80 80 75 75 70 70 65 65 11.57 55 50 In te n s ity , c p s In te n s ity , c p s 60 11.55 60 55 50 11.53 11.53 11.60 45 45 11.60 11.61 40 40

35 35 30 30 25 25 11.50 20 11.64 20 15 15 11.52 11.66 10 10 5 5 0 11.64 11.30 11.32 11.34 11.36 11.38 11.40 11.42 11.44 11.46 11.48 11.50 11.52 Time, min 25 ms MRM; 22% RSD 11.54 11.56 11.58 0 11.60 6.5 11.62 11.647.0 11.66 11.87 11.68 7.5 11.70 11.72 8.0 11.74

8.5 9.0 9.5 10.0 Time, min peak definitions troubles 10.5 11.0 11.5 12.24 12.0 12.64 12.5 Impact of dwell time: SIM vs MRM Sample Name: "10 ms SIM" Sample ID: "" File: "03032009SET1.wiff" Peak Name: "1012.4 M+14" Mass(es): "1012.4 amu" Comment: "" Annotation: "" Sample Index: 29 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.000 ng/mL 1.20e4 Acq. Date: 3/3/2009 Acq. Time: Sample Name: "10 ms MRM" Sample ID: "" File: "03032009SET1.wiff" Peak Name: "1012.3 / 147.2" Mass(es): "1012.4/147.1 amu" Comment: "" Annotation: "" Sample Index: 57 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.000 ng/mL 195 Acq. Date: 3/4/2009 Acq. Time: 11:46:50 AM 190 Modified: Yes 185 Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 1.45 cps 180 Area Threshold: 7.27 cps Num. Smooths: 3 175 Sep. Width: 0.20 Sep. Height: 0.01 170 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 165 Exp. Val. Ratio: 3.00 RT Window: 80.000 sec Expected RT: 11.548 min 160

Use Relative RT: No 9:49:49 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 56.13 cps Area Threshold: 280.65 cps Num. Smooths: 3 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.000 sec Expected RT: 11.591 min Use Relative RT: No 1.15e4 1.10e4 1.05e4 1.00e4 Int. Type: Manual Retention Time: 11.561 min Area: 1.991e+004 counts Height: 4.540e+003 cps Start Time: 11.359 min End Time: 11.699 min 9500.00 11.60 12.35 155 Int. Type: Base To Base Retention Time: 11.548 min Area: 5.516e+001 counts Height: 1.960e+002 cps Start Time: 11.540 min End Time: 11.554 min 9000.00 11.55 150 145 140 8500.00 135 130 8000.00 125 7500.00 120 115 7000.00 11.56

110 Intensity, cps Intensity, cps 6500.00 6000.00 105 100 95 11.61 90 11.53 5500.00 11.60 85 80 5000.00 75 4500.00 11.51 9.96 3500.00 8.51 8.57 8.36 3000.00 8.31 2500.00 8.45 8.13 7.88 2000.00 8.72 8.74 9.06 9.15 8.85 10.95 10.14 9.61 12.28 12.57 12.6165 10.74 10.07 10.38 9.69 10.48 10.60 11.41 11.84 11.89 12.46 10.70 11.44 12.27 6012.7412.78

55 11.17 9.729.93 9.41 12.22 11.77 10.84 9.52 70 12.06 10.01 4000.00 12.89 50 11.24 9.28 45 40 8.97 35 8.23 30 25 1500.00 20 15 1000.00 10 500.00 5 0 0.00 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 6.5 7.0 13.0 7.5 8.0

8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 Time, min Time, min 10 ms SIM; 11% RSD 10 ms MRM; 53% RSD Fused-Core Ascentis Express Particles Comparison of architecture of FusedCore particles vs. porous 3 m particles 27 2x50 Polaris C18 3m 180 A versus 2x30 Ascentis Express Peptide 160A XIC of +Q1 MI (4 ions): 1007.7 amu from Sample 40 (Fresh solvent 4.9 -5.7 35 ul) of 07062010SET1.wiff (Turbo Spray) Max. 9.7e4 cps. 11.50 9.5e4 9.0e4 8.5e4 S/N 170 8.0e4 7.5e4 7.0e4 6.5e4 6.0e4 I n t e n s it y , c p s 5.5e4 S/N 40 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4 5000.0 0.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 Time, min 10.0 10.5 11.0

11.5 12.0 12.5 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 m ID PEEKsil vs. 100 m PEEK 100 m ID PEEKsil vs. 125 m PEEK XIC of +Q1 MI (4 ions): 1007.7 amu from Sample 16 (red tube, no valve) of 11122010SET1.wiff (Turbo Spray) Max. 2.6e4 cps. 3.0e4 4.05l vs 6.35l 2.8e4 2.6e4 11.90 2.4e4 20% S/N gain 2.2e4 2.0e4 1.8e4 I n t e n s ity , c p s 1.6e4 1.4e4 1.2e4 1.0e4 8000.0 6000.0 10.52 4000.0 10.46 2000.0 0.0 6.5 7.0 7.5 8.0 8.5 9.0 11.30 11.62 10.36 9.00 6.0 10.69 9.5 Time, min 10.0 10.5 11.0

11.5 12.03 12.0 12.75 12.5 TTR Mx/M+0 C-peptide d -L eu ciGn ild e n c o r p o r a t i o n i n t o C p e p t i d e u r g H y p e r g l y c e m i a d L e u c i n e M e a l u c o s e 250m g /d l M 1 L e u c i n e 2

0.08 0.06 0.04 0.02 0.00 -60 T im e(p o stracerin tro d u ctio n ) 0 60 120 180 240 300 360 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 m] allows large sample volume to be injected Use of PEEKSIL tubing improves S/N Acknowledgements Funding: NIH: MO1-RR12248 NIH: R01 DK61644-01 Technical Support: Agilent Technologies Applied Biosystems Phenomenex Supelco SGE

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