Preanalytical Variability and the Stability of Human Plasma Proteins:
Mass Spectrometry Changes After Blood Collection
J. Yi, H.H. Quek, T. Finocchio, and C.A. Gelfand
BD Diagnostics, Franklin Lakes, New Jersey, USA
Introduction
Abstract
Human plasma and serum contain a wide variety of
proteins, which hold the potential of being biomarkers
for proteomics-based diagnostic and prognostic assays.
These proteins, however, are subject to degradation
and modification both during and after blood
collection. The aims of this study are to investigate
the stability of plasma and serum samples, and to
establish a recommendation for a standard procedure
for sample collection and storage for proteomic
studies. The effects of anticoagulants, protease
inhibitors, blood clotting, and length of storage time
have been investigated. Peptides (750-3200 Da) in
samples subjected to a range of preanalytical variables
were extracted using cut-off membrane filters and
monitored with Matrix-Assisted Laser Desorption
Ionization Time-of-Flight Mass Spectrometry (MALDITOF MS). Our observations indicated that peptide
mass spectra of plasma differ significantly from those
of serum. The identification of peptides uniquely
observed in serum suggests that they are generated
from fibrinogen due to blood clotting. Differences in
peptides are observed among plasma collected using
different anticoagulants, including EDTA, sodium
citrate, and heparin, consistent with the different
anticoagulant effects relative to the clotting cascade.
Sequences of these peptides have also been identified,
and their biological significance will be discussed.
Further studies also indicate that plasma proteins
are subject to proteolytic digestions during and after
blood collection in a time dependent manner. Protease
inhibitors mixed immediately with blood in a blood
collection tube during phlebotomy provide enhanced
benefits for stabilization of blood proteins.
Human plasma and serum are extremely complex
protein-containing samples1. Complexities of such
samples include:
• Wide dynamic range in protein abundance
• The 20 most prevalent proteins account for more
than 95% of the protein mass
• Intrinsic blood proteins are comingled with
proteins secreted from other cells and tissues.
• Blood also contains foreign proteins from
infectious organisms or parasites.
• Heterogeneity of post-translational modifications
• Peptides generated from fragmentation of
proteins
• Potential effects of anticoagulants on peptidome
and proteome
Handling of blood samples is common in modern
medicine. Given the richness of blood’s information
content, it has become an attractive target for protein
biomarker discovery and holds the promise of a
breakthrough in disease diagnosis and therapeutic
monitoring.1-3
2. Sample preparations for MALDI-TOF MS4 : Serum
and plasma samples, either from EDTA-only
control tube or BD™ P100 with PIs, were incubated
simultaneously at room temperature. At the
specified time, a 20-50 µl aliquot was withdrawn
and any proteolytic reactions were quenched by
adding 2% triflouroacetic acid (TFA) up to 0.1% (v/
v) final concentration. The quenched samples were
loaded to a Microcon YM-3 (Millipore) and spun in
a Micro Centrifuge (Eppendorf™ Centrifuge 5417R)
under 13,000 rpm for 30 minutes. The filtrate was
collected and desalted using ZipTip® C18 (Millipore).
The peptides (1 µL) eluted from ZipTip® was mixed in
1:1 ratio with a matrix solution (5 mg/mL of
α-cyano-4-hydroxycynnamic acid, 60% acetonitrile,
0.1% (v/v) TFA and 1 mM monobasic ammonium
phosphate) and spotted on a plate for MALDI-TOF
MS analysis.
3. MALDI-TOF MS and TOF/TOF MS: The MALDI-TOF
MS and MS/MS analysis were performed on a Ultraflex
II (Bruker Daltonics). MS spectra were recorded in
the positive ion reflector mode with 2000 laser shots,
using internal calibration setting. The mass range was
collected from 800 to 3200 M/z. For MALDI MS/MS
analysis, all precursors of interest were selected for MS/
MS fragmentation in order to identify the peptides. All
the MS/MS data was acquired using the default 1 kV
MS/MS mode of the Ultraflex II. The default calibration
parameters of the instrument and MALDI plate were
updated daily in both MS and MS/MS operating modes.
During MS/MS data acquisition, the timed-ion-selector
(TIS) window was set to a width of 10 Da. The MS/
MS spectra were processed in either FlexAnalysis or
ClinProTools (Bruker Daltonics), and the peak list was
exported into a Micromass (.PKL) format for database
searching against the NCBInr protein sequence database
using Mascot (Matrix Science, London, UK).
Plasma and serum peptides can be altered ex vivo
by many biochemical pathways, including proteolysis,
phosphorylation, glycosylation, and other posttranslational modifications. Digestion of proteins by
intrinsic plasma proteases, during the processes of
blood collection and subsequent sample preparation,
is often an overlooked variable. Here, we present a
study of variability of sample specimens caused by
anticoagulants and show that the clotting process
generates many peptides in serum. Plasma collected
with different anti-coagulants also give different
peptide specimen.
Proteolytic degradation of human plasma was
investigated by MALDI-TOF peptide mass analysis after
a time-course incubation. We demonstrate that plasma
and serum proteins can be significantly affected by
proteolytic digestion, whereas inclusion of protease
inhibitors (PIs) in the blood collection tube (BD™ P100)
mitigates this effect. The data indicate that immediate
exposure of blood to PIs, included in an evacuated
tube, helps to preserve MS profiles by reducing the
amount of proteolytic activity.
Methods and Procedures
1. Blood collection and plasma/serum preparations:
Human blood was directly drawn into evacuated
tubes, including glass serum tubes (red top) and
plasma tubes containing anticoagulants: buffered
citrate, Lithium heparin, EDTA or BD™ P100 tubes
(EDTA plus protease inhibitors). After collection
of the blood specimen, the serum blood samples
were allowed to clot for 60 minutes followed by
centrifugation at 2,500xg for 15 minutes at room
temperature. Plasma blood samples were spun at
2,500xg for 15 minutes at room temperature. The
EDTA contained plasma was separated from cells by
a mechanical separator (Figure 1), removed from
the blood collection tubes and transferred into
Eppendorf™ tubes for either immediate MS analysis
or storage at -80ºC until next usage.
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Results
EDTA plasma versus Serum
• Both EDTA plasma and serum were collected from 8 healthy individuals.
• Blood from 8 subjects was collected into EDTA plasma tubes and glass
serum tubes (red top).
• Sera were obtained by incubation of whole blood for 60 minutes to
allow for complete clotting before centrifugation.
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• As we would expect, unique variations can be
seen across 8 individuals, which may suggest a
source potential biomarkers
• More peptides were found in serum than in
EDTA plasma:
– Suggesting more proteolytic digestions of proteome
in sera
– Clotting process causes more peptides in MALDI-TOF MS
– Peptide sequence by MS/MS sequence revealed most
of these peptides are from fibrinogen
• EDTA plasma provides a more intact specimen
for proteomics-based analyses.
Proteolytic Degradation
in Plasma and Serum
Enhanced stability over time
provided by PI cocktail
• Time-course incubation of the plasma and sera
at room temperature:
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Effects of Anticoagulants
– More peptides in heparin plasma were observed than
in citrate plasma, from 1500-3000 Da (M/z)
– Suggests that citrate preserves plasma protein better
than heparin
• Different anticoagulants lead to different
peptide profiles, suggesting each may have a
unique effect on certain plasma proteins.
• The mechanistic causes of these differences
are under investigation.
EDTA plasma versus BDTM P100 plasma
• Blood from 8 subjects was collected into EDTA
plasma tubes and BD™ P100 tubes
• BD™ P100 tube is a EDTA tube with additional
protease inhibitors
• BD™ P100 plasma also indicates variants among
individuals
• Addition of protease inhibitors does not
alter the effect of EDTA anticoagulation,
as interpreted by the peptide profiles shown.
• Several peptides at ~ 1100 Da (m/z) observed in
EDTA plasma were not found in P100 plasma.
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• Proteolytic degradations were observed in all plasma
and serum samples
• Changes in peak identities and intensities can be seen as
evolution of new peptides over time, and also in loss of
peptides present initially
• Peptide changes were found to be more intense in
serum than in plasma
• Comparing EDTA, citrate, and heparin plasma, heparin
plasma appears to have the most proteolytic degradation
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• Variation among individuals was observed from
both heparin and citrate plasma.
• Different peptide spectra were found between
heparin plasma and citrate plasma:
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• Samples were quenched by addition of TFA
up to 0.1 % to stop any further chemical
modification of proteins and peptides
• Peptides were extracted from quenched
samples for MALDI-TOF MS analysis
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Heparin plasma versus Citrate plasma
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– Time 0 was defined to be no incubation after plasma
or sera were obtained
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• Both EDTA and P100 samples were acquired
within 15 minutes and processed under the
same conditions
• Peak heights at 1165.7, 1211.7, 1739.9, 1762.0,
1847.1, and 1859.9 are roughly equivalent in
two spectra
• Apparent inhibition of ex-vivo peptide
generation by PIs*:
• Each averaged spectrum was from
10 individuals
• By comparison of EDTA and P100 spectra at
both time 0 and 5 hours, the stabilization
benefit provided by P100 was observed:
– A greater number of smaller peptides were observed
in EDTA than in P100 plasma, in terms of both peptide
number and abundance
– Larger peptides are more abundant in P100 than
in EDTA plasma
• After 5 hours of incubation, P100 plasma
profile was found to be less changed than
EDTA plasma.
– Peaks at 1060.6 and 1076.6 are observed in EDTA, but not
in P100, and peak 1098.6 in EDTA is higher than in P100;
• Preservation of intrinsic plasma peptides*:
– Peaks at 1778.0 and 1865.0 are higher in P100 than in
EDTA, suggesting that P100 enhances their stability.
• Stabilization provided at “Time Zero” suggests the
importance of immediate exposure of blood to PIs.
*These observations are supported by time-course studies
Conclusions
References:
• Peptide profiles of both serum and plasma were shown to be:
– Variable among individuals
– Changed by intrinsic proteolysis
• Peptides change more intensely in serum than in plasma
• EDTA and citrate plasma are more stable than heparinized plasma
• BDTM P100 maintains the anticoagulant activity of EDTA and provides enhanced stability of
peptides and proteins compared to EDTA plasma
1. Anderson NL, Anderson NG. The human plasma proteome: history, character, and diagnostic prospects.
Mol Cell Proteomics. 2002; 1: 845-867.
2. Petricon E, Wulfkuhle J, Espina V, Liotta LA. Clinical proteomics: revolutionizing disease detection and patient
tailoring therapy. J Proteome Res. 2004; 3:209-217.
3. Rai AJ, Chan DW. Cancer proteomics: serum diagnostics for tumor marker discovery. Ann NY Acad Sci. 2004;
1022: 286-294.
4. Yi J, Skalka AM. Mapping epitopes of monoclonal antibodies against HIV-1 integrase with limited proteolysis and
matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Biopolymers. 2000; 55:308-318.
www.bd.com/proteomics
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Preanalytical Variability and the Stability of Human Plasma Proteins