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References
1. Krastins, B.; Prakash, A.; Sarracino, D. A.; Nedelkov,
D.; Niederkofler, E. E.; Kiernan, U. A.; Nelson, R.;
Vogelsang, M. S.; Vadali, G.; Garces, A.; Sutton, J. N.;
Petermant, S. M.; Byram, G.; Darbouret, B.; Perusse,
J. R.; Seidah, N. G.; Coulombe, B.; Gobom, J.; Portelius,
E.; Pannee, J.; Blennow, K.; Kulasingam, V.; Couchman,
L.; Moniz, C.; Lopez, M. F. Rapid development of
sensitive, high-throughput, quantitative and highly
selective mass spectrometric targeted immunoassays for
clinically important proteins in human plasma and
serum. Clinical Biochemistry 2013, 46 (6), 399-410.
2. Niederkofler, E. E.; Phillips, D. A.; Nedelkov, D.;
Kiernan, U. A.; Krastins, B.; Peterman, S.; Garces, A.;
Lopez, M. F. A universal mass spectrometric
immunoassay (MSIA) model system based on human
insulin-like growth factor 1 (IGF1). In Application
Notes, Scientific, T. F., Ed. 2013; Vol. 1001.
3. Oran, P. E.; Jarvis, J.W.; Borges, C.R.; Sherma, N. D.;
Nelson, R. W. Mass spectrometric immunoassay of
intact insulin and related variants for population
proteomcis studies. Proteomics Clinical Applications
2011, 5 (7-8), 454-459.
4. Couchman, L.; Taylor, D. R.; Moniz, C. F.; Niederkofler,
E. E.; Ribar, A.; Antwi, K.; Kiernan, U. A.; Krastins, B.;
Peterman, S. MSIA insulin workflow for the
comprehensive identification and analysis of isobaric
insulins: a method for insulin mixture quantitation
using HR/AM and mulitplexed LC. Scientific, T. F., Ed.
2014; Vol. 0614.
5. Peterman, S. M.; Niederkofler, E. E.; Phillips, D. A.;
Krastins, B.; Kiernan, U. A.; Tubbs, K., A.; Nedelkov, D.;
Prakash, A.; Vogelsang, M. S.; Schroeder, T.; Couchman,
L.; Taylor, D. R.; Moniz, C. F.; Vadali, G.; Bryam, G.;
Lopez, M. F. An automated, high-throughput method
for targeted quantification of intact insulin and its
therapeutic analogs in human serum or plasma coupling
mass spectrometric immunoassay with high resolution
and accurate mass detection (MSIA-HR/AM).
Proteomics 2014 , 14 (12), 1445-1456.
For research use only. Not for use in diagnostic procedures.
Conclusion
An automated, high-throughput data processing workflow
for detection and targeted quantification of insulin and its
analogs at low levels in biological matrices was presented
for use in research applications.
Full-scan MS data could be analyzed due to the low noise
and selectivity of the MSIA extraction technology.
Co-eluting insulin analogs were easily separated and
identified based on the accurate m/z values of each
precursor charge state and corresponding isotopes.
Detection and quantification ranges were 1.5 to 960 pM
in a plasma matrix. Even at low analyte levels, the
quality of the quantitation was high. Linear regression
values for the method were all better than 0.98 using
a 1/x-weighting scheme. The average dot product
coefficients for the porcine insulin ISTD were consistently
above 0.97 at the 50 pM level. Though the lowest four
levels used in the study were 50–100 times lower than
previously published, %RSD values were acceptably low.
The HRAM data acquisition capabilities of the
Q Exactive mass spectrometer enabled streamlined
qualitative and quantitative data processing and reporting
using Pinpoint software. This approach enabled
quantification of HRAM MS data using the precursor
charge state distribution as well as the isotopic
distribution for evaluation of potential background
interference. By acquiring data in a nontargeted manner,
post-acquisition methods can be used to process the data
later for any insulin variant sequence or modification.
Data processing was performed in 30 minutes using a
single Pinpoint software workbook containing all of the
targeted insulin variants. To achieve robust quantitation,
the Pinpoint software data processing method used the
precursor charge state distribution and isotopic
distribution analysis for evaluation of potential
background interference. Pinpoint software capabilities,
such as color-coding capability and the Main Workbook,
which consolidates qualitative and quantitative tables and
graphs in an interactive display, facilitated data review.
AN64279-EN 1214S
Application Note 619
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