LC-MS analysis of plasma from patients

with Insulin Autoimmune Syndrome.

Explaining spurious immunoassay results.

Dr Richard Kay

Metabolic Research Laboratories

Cambridge University

Overview

• Brief introduction to Insulin

- How is it made?

- Insulin Autoimmune Syndrome (IAS) / Hirata’s disease

• LC-MS analysis of insulin peptides in plasma

- Techniques involved (targeted and untargeted approaches)

• Comparison of LC-MS and Immunoassay data

• GFC-LC-MS and SEC-LC-MS

Proinsulin processing in pancreatic -cells

Insulin Autoimmune Syndrome (IAS)

a.k.a. Hirata’s disease

• Patients develop antibodies against insulin

- Autoimmune disease

- Essentially similar to an ADA response to a peptide drug

• Plasma insulin concentrations are significantly raised

- Sudden increase in plasma insulin levels causes hypoglycaemia

• Diagnosis is based on insulin to C-peptide ratios

- But, C-peptide and insulin ELISA results can give spurious results

• Have to rule out insulin poisoning..

- Presence of anti-insulin antibodies further helps diagnosis

Insulin and insulin auto-antibodies:

Issues with immunoassays

Capture Ab

Detection Ab

Normal ELISA process in control plasma

Insulin and insulin auto-antibodies:

Issues with immunoassays

Immunoassay / Immunocapture

reagents

Anti-insulin antibodies

Normal ELISA process in IAS plasma

Can LC-MS be used as an alternative?

Plasma peptide LC-MS analysis strategy

• Plasma protein precipitation with 80% acetonitrile, 20% water

• Small amount of water slows the precipitation event

- Significantly increases peptide recovery

- Disrupts peptide-Ab interactions

- Precipitates Ab

• Transfer supernatant, evaporate and perform SPE

- Removes highly hydrophobic contaminants (phospholipids etc.)

• Perform nano or high flow LC-MS analysis

Endogenous and exogenous insulin LC-MS analysis

STD 8

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

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181102-1011 2: MRM of 3 Channels ES+ 1184 > 1180.66 (Detemir intact 3)

1.95e67.07

181102-1011 Sm (Mn, 3x1) 1: MRM of 21 Channels ES+ 1165.96 > 219.1 (Aspart low mass)

3.03e54.41

5.174.84

181102-1011 Sm (Mn, 3x1) 1: MRM of 21 Channels ES+ 1162.2 > 226.29 (Human insulin 3)

9.93e54.61

4.47

181102-1011 Sm (Mn, 3x1) 1: MRM of 21 Channels ES+ 1162.2 > 216.88 (Humalog intact 2)

1.19e64.47

181102-1011 Sm (Mn, 3x1) 1: MRM of 21 Channels ES+ 1011 > 1179 (Glargine 1)

2.31e43.29

3.623.704.38 5.81

181102-1011 Sm (Mn, 3x1) 1: MRM of 21 Channels ES+ 1007.37 > 1047.1 (Human C-peptide 3)

1.55e52.82

181102-1011 Sm (Mn, 3x1) 1: MRM of 21 Channels ES+ 956.3 > 1120.8 (Bovine Insulin IS intact)

1.50e44.09

• LC-MS/MS can differentiate between

insulin and analogues

• Example data from solvent crash and

SPE extraction approach

• Can also measure C-peptide

• Immunoprecipitation based insulin

methods wouldn’t capture C-peptide

Detemir

Aspart

Actrapid

Humalog

Glargine

C-peptide

Bovine insulin

Insulin measurements (LC-MS Vs Immunoassay)

Absolute quantitation

High flow analyses

R2=0.974, slope = 0.87 R2=0.984

Relative quantitation

Nanoflow analyses (2hr method)

Immunoassay Vs targeted LC-MS in IAS

Combined insulin and C-peptide measurements

InsulinInsulin immunoassay

tends to under report

Insulin LC-MS Vs ELISA – good correlation for controls, but poor for IAS subjects..

C-peptide

C-peptide immunoassay tends

to severely over report

C-peptide LC-MS Vs ELISA – good correlation for controls, but poor for IAS subjects..

What is causing this discrepancy?

Plasma peptidomics results: Controls Vs IAS

Control sample

Insulin autoimmune syndrome patient

B-chain C-peptide A-chain

Multiple peptides spanning

proinsulin cleavage sites

What are the real

circulating peptides?

Intact insulin precursor molecule MS/MS analysis:

Des 31-32 proinsulin product ion spectrum

Precursor m/z = 1300, z=7

Precursor m/z = 1281.62, z=7

des 31-33

proinsulin

Intact insulin precursor molecule MS/MS analysis:

Des 31-33 proinsulin product ion spectrum

Precursor m/z = 1342.00, z=7

Proinsulin standard

Intact insulin precursor molecule MS/MS analysis:

Proinsulin product ion spectrum

Cause of incorrect C-peptide measurement?

Normal plasma

+ELISA Ab’s

ELISA detects C-peptide

in des 31-32 proinsulin

Bound insulin’s take on the

Ab half life!

CaptureDetection

Hirata’s disease plasma

ELISA Ab’s

Gel filtration chromatography separations

C-peptide and Des-31-32 proinsulin (MS Vs ELISA)

C-p

eptide E

LIS

A

C-peptide elution

IgG elution time

C-peptide elution

C-p

eptide L

C-M

S

Recent tests show C-peptide immunoassay

cross reacts 100% with proinsulin

Pro

insulin

sLC

-MS

LC-MS can differentiate between all

proinsulin processing products

GFC (500µL plasma) Vs SEC (1 µL plasma)

706

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190226-005 Sm (Mn, 1x1) MRM of 21 Channels ES+ 1162.5 > 226.15 (Human insulin 3)

3.86e714.78

15.23

190226-005 Sm (Mn, 1x1) MRM of 21 Channels ES+ 1342.08 > 219.14 (Proinsulin 1)

8.21e414.55

18.6616.9814.98

190226-005 Sm (Mn, 1x1) MRM of 21 Channels ES+ 1300 > 1523.33 (Des 31-32 proinsulin 3)

1.01e614.79

15.23

190226-005 Sm (Mn, 1x1) MRM of 21 Channels ES+ 1281.62 > 1523.33 (Des 31-33 proinsulin)

9.31e418.3314.75

18.21

18.1115.19

18.43

18.69

190226-005 Sm (Mn, 1x1) MRM of 21 Channels ES+ 956.3 > 1120.8 (Bovine insulin)

4.51e514.60

14.74

GFC data, 500 µL plasma (3 hour runtime)

Insulin

Proinsulin

Des 31-32

Des 31-33

Bovine insulin

Microflow LC-MS analysis at 1 µL/minute

TQ-XS with nanospray interface

Sample 3.5min 1

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190412-047 Sm (Mn, 1x1) 2: MRM of 17 Channels ES+ 1162.5 > 226.15 (Human insulin 3)

1.87e512.27

18.68

16.5815.79

13.00

18.42

26.40

190412-047 Sm (Mn, 1x1) 2: MRM of 17 Channels ES+ 1342.08 > 219.14 (Proinsulin 1)

2.12e416.14

16.04

15.85

15.71

15.258.101.27

8.55 14.229.32 12.18

16.18

18.76

16.69

17.27 18.84

26.3919.3123.6320.94

190412-047 Sm (Mn, 3x1); Sm (Mn, 2x1); Sm (Mn, 1x1) 2: MRM of 17 Channels ES+ 1300 > 1523.33 (Des 31-32 proinsulin 3)

1.17e412.33

18.7512.94

190412-047 Sm (Mn, 1x1) 2: MRM of 17 Channels ES+ 1281.62 > 1523.33 (Des 31-33 proinsulin)

7.43e315.96

15.82

12.28

16.14

16.39

18.6916.63

190412-047 Sm (Mn, 1x1) 2: MRM of 17 Channels ES+ 956.3 > 1120.8 (Bovine insulin)

8.73e511.99

SEC data, 1 µL plasma (10 minute runtime)

Insulin

Proinsulin

Des 31-32

Des 31-33

Bovine insulin

Loss of bovine insulin IS via adsorption to consumables

Protein elution

Quan recovery plate

Other Lobind plate

Conclusions

• IAS plasma causes serious issues with existing immunoassays

• Plasma peptidomics analysis identified incomplete insulin processing products

• Intact LC-MS analysis confirmed presence of Proinsulin and Des 31-32 proinsulin

- Both GFC and SEC separation prior to LC-MS also confirmed these as antibody bound

• Des 31-32 severely cross reacts with C-peptide ELISA

- Potential new biomarker to distinguish between IAS, insulin poisoning and other insulin diseases.

• LC-MS is well suited to the analysis of proinsulin derived products in plasma

- Not for routine sample analysis – only for “funnies”

Acknowledgements

• Cambridge University

- Professors Fiona Gribble and Frank Reimann

- Dr. Geoff Roberts, Dr. Pierre Larraufie, Rachel Foreman

• Addenbrookes Hospital

- Dr. David Church, David Halsall

• Waters

Thanks for your attention!

Any questions?