Targeted On-line SPE-LC-MS/MS Assay for the Quantitation
of 12 Apolipoproteins from Human Blood
Julia Dittrich,* Melanie Adam, Hilke Maas, Max Hecht, Madlen Reinicke,
L. Renee Ruhaak, Christa Cobbaert, Christoph Engel, Kerstin Wirkner, Markus L
Joachim Thiery, and Uta Ceglarek
Laborious sample pretreatment of biological samples represents the most
limiting factor for the translation of targeted proteomics assays from research
to clinical routine. An optimized method for the simultaneous quantitation of
12 major apolipoproteins (apos) combining on-line SPE and fast LC-MS/MS
analysis in 6.5 min total run time was developed, reducing the manual sample
pretreatment time of 3 μL serum or plasma by 60%. Within-run and
between-day imprecisions below 10 and 15% (n = 10) and high recovery rates
(94–131%) were obtained applying the high-throughput setup. High-quality
porcine trypsin was used, which outperformed cost-eﬀective bovine trypsin
regarding digestion eﬃciency. Comparisons with immunoassays and another
LC-MS/MS assay demonstrated good correlation (Pearson’s R: 0.81–0.98).
Further, requirements on sample quality concerning sampling, processing,
and long-term storage up to 1 year were investigated revealing signiﬁcant
inﬂuences of the applied sampling material and coagulant on quantitation
results. Apo proﬁles of 1339 subjects of the LIFE-Adult-Study were associated
with lifestyle and physiological parameters as well as establish parameters of
lipid metabolism (e.g., triglycerides, cholesterol). Besides gender eﬀects, most
signiﬁcant impact was seen regarding lipid-lowering medication. In
conclusion, this novel highly standardized, high-throughput targeted
proteomics assay utilizes a fast, simultaneous analysis of 12 apos from least
J. Dittrich, M. Adam, H. Maas, M. Hecht, M. Reinicke, Prof. Dr. J. Thiery,
Prof. Dr. U. Ceglarek
Institute of Laboratory Medicine, Clinical Chemistry and Molecular
University Hospital Leipzig
J. Dittrich, Dr. C. Engel, Dr. K. Wirkner, Prof. Dr. M. L
oﬄer, Prof. Dr. J.
Thiery, Prof. Dr. U. Ceglarek
LIFE, Leipzig Research Center for Civilization Diseases
Department of Clinical Chemistry and Laboratory Medicine
Leiden University Medical Center
Leiden, The Netherlands
Dr. C. Engel, Prof. Dr. M. L
Institute for Medical Informatics, Statistics and Epidemiology
Due to their central role in lipid
metabolism, apolipoproteins (apos) are
potential prognostic biomarkers for dis-
orders of lipoprotein metabolism that are
highly correlated with the development
of cardiovascular disease.
validated clinical immunoassays are
expensive and only available for the
quantitation of the established cardio-
vascular risk factors apoA-I and apoB.
The implementation of LC-MS/MS
into quantitative proteomics research
represented an analytical revolution
since targeted proteomics assays allow
for the multiplexed analysis of proteins
via enzymatically formed signature
In contrast to functional investigations
that mostly rely on comparisons of rel-
ative quantities of hundreds of proteins,
absolute quantiﬁcation of selected pro-
teins is required for clinical application.
Biomarker discovery studies use complex
software-assisted label-free techniques
for protein quantitation,
isotope-labeled (SIL) internal standards
are applied for quantiﬁcation in clinical assays.
three diﬀerent forms of internal standards are distinguished:
SIL peptides, SIL surrogates including enzymatic cleavage sites
and recombinant SIL full-length proteins.
Due to cost eﬀec-
tiveness and limited availability of SIL protein standards, SIL
peptides are predominantly used in targeted proteomics assays.
However, sample preparation procedures have to be highly stan-
dardized especially concerning digestion to ensure a complete
formation of signature peptides from endogenous proteins.
The selection of such quantotypic peptides is subject to a num-
ber of strict criteria.
Besides omission of post-translational
modiﬁcations and polymorphisms, also the expulsion of the ox-
idizable amino acids methionine and cysteine is recommended.
Due to the limited number of tryptically formed peptides, espe-
cially latter restriction is problematical for the analysis of smaller
proteins like apoM. However, a standardized and reproducible
alkylation of thiols, which is besides protein denaturation, reduc-
tion of disulﬁde bonds, and digestion an integral part of targeted
proteomics workﬂows, oﬀers the opportunity to use cysteine-
Proteomics 2018, 1700279
2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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