乳清蛋白分析干扰评估：液相色谱-串联质谱法研究
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"液相色谱-串联质谱法在乳清蛋白分析中评估了牛α-乳白蛋白肽与其同位素标记肽之间的相互干扰情况。该研究发表在《色谱A》期刊2018年第1533期，页码94-101。文章探讨了在使用液相色谱-串联质谱(LC-MS/MS)技术进行乳清蛋白分析时，牛α-乳白蛋白肽与同位素标记肽可能存在的相互影响，这对于提高蛋白质组学研究的准确性和可靠性至关重要。"
在这篇研究论文中，作者Zhen Wang、Qi Chen、Qiong Wu、Qifeng Liu、Da Chen、Xiaogang Chu等人来自中国天津大学精密仪器与光电子工程学院、中国检验检疫科学研究院以及黄山出入境检验检疫局，他们在2017年10月26日提交了原始稿件，并在同年12月2日进行了修订，最终在12月9日被接受并在线发布。
关键词包括“液相色谱-串联质谱法”、“牛α-乳白蛋白肽”、“内标”、“干扰效应”。文章的重点在于内标的使用，特别是同位素标记肽，以减少分析过程中可能出现的误差。在蛋白质组学研究中，内标通常用于定量分析，以补偿样品处理、仪器变化和测量过程中的不确定性。然而，当内标与目标肽段之间存在相互干扰时，这可能导致分析结果的偏差。
LC-MS/MS技术是蛋白质组学研究中常用的一种方法，它结合了液相色谱的分离能力和质谱的高灵敏度检测，能够精确地鉴定和量化蛋白质或其肽段。在本研究中，作者评估了牛α-乳白蛋白肽（一种存在于乳清蛋白中的重要成分）与它的同位素标记形式之间的相互作用，以确定这种干扰是否会影响分析的准确性。这种评估对于优化分析策略，特别是在复杂生物样本中精确测定特定蛋白质水平，具有重要意义。
通过实验和数据分析，研究人员可能已经确定了最佳条件，以最小化这种潜在的干扰，从而提供更可靠的结果。这些发现对于食品科学、生物医学研究和临床诊断等领域具有实际应用价值，特别是在检测和量化食品中的乳蛋白或与乳蛋白相关的生物标志物时。此外，这项工作也强调了在设计和解释LC-MS/MS实验时，考虑并控制相互干扰的重要性。
Journal
of
Chromatography
A,
1533
(2018)
94–101
Contents
lists
available
at
ScienceDirect
Journal
of
Chromatography
A
jo
ur
nal
ho
me
pag
e:
www.elsevier.com/locate/chroma
Evaluation
of
mutual
interference
between
bovine
␣-lactalbumin
peptide
and
its
isotope-labeled
peptide
in
whey
protein
analysis
using
liquid
chromatography-tandem
mass
spectrometry
Zhen
Wang
a,b
,
Qi
Chen
c
,
Qiong
Wu
c
,
Qifeng
Li
a
,
Da
Chen
a,∗
,
Xiaogang
Chu
b,∗
a
College
of
Precision
Instrument
and
Opto-Electronics
Engineering,
Tianjin
University,
Tianjin,
300072,
PR
China
b
Chinese
Academy
of
Inspection
and
Quarantine,
Beijing,
100176,
PR
China
c
Huangshan
Entry-Exit
Inspection
and
Quarantine
Bureau,
Huangshan,
230022,
PR
China
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
26
October
2017
Received
in
revised
form
2
December
2017
Accepted
9
December
2017
Available
online
9
December
2017
Keywords:
Liquid
chromatography-tandem
mass
spectrometry
Bovine
␣-lactalbumin
peptide
Internal
standard
Interference
effect
a
b
s
t
r
a
c
t
Internal
standard
(IS)
method
is
commonly
used
to
correct
the
matrix
effect
of
samples
in
the
liquid
chromatography-tandem
mass
spectrometric
(LC–MS/MS)
analysis
of
whey
proteins.
However,
the
pres-
ence
of
mutual
interference
between
some
peptides
and
their
isotope-labeled
peptides
distorts
the
MS
signals,
requiring
a
fundamental
evaluation
to
understand
the
phenomenon
of
signal
variations.
In
this
study,
a
simple
strategy
is
proposed
to
evaluate
the
effects
of
sample
pretreatment,
materials
and
dilu-
tion
of
solutions
on
the
MS
signals
of
␣-lactalbumin
(VGINYWLAHK)
and
-lactoglobulin
peptides
using
two
typical
LC–MS/MS
systems,
Q-Trap
and
Q-Orbitrap.
The
strategy
adapts
the
experimental
MS
data
to
optimize
methods,
thus
providing
meaningful
solutions
to
suppress
the
mutual
interference
presented
in
the
analysis
of
peptides.
As
a
result,
the
strategy
through
the
combination
of
100-fold
dilution
and
plastic
injection
vial
improves
the
quantitation
results
of
␣-lactalbumin
peptide
significantly.
While
the
-lactoglobulin
peptide
presents
different
phenomenon
of
signal
variations
when
compared
with
that
of
␣-lactalbumin
peptide,
revealing
that
each
peptide
needs
to
be
optimized
individually.
The
calibration
effect
of
different
IS
was
also
studied
in
fifteen
infant
milk
powders
to
confirm
the
mutual
interference
impact
to
quantification
result.
These
results
indicated
that
a
simple
strategy
through
the
combination
of
sample
dilution
and
plastic
injection
vial
could
be
well
extended
to
quantitative
analysis
of
any
other
peptide
in
the
complex
systems.
©
2017
Published
by
Elsevier
B.V.
1.
Introduction
Alpha-lactalbumin
(␣-lactalbumin)
reperesents
the
dominant
protein
in
human
milk
that
comprises
25–35%
of
the
total
pro-
tein
content
[1].
It
primarily
contributes
to
the
neurodevelopment,
regulating
sleep
and
mood
of
infants
[2–4].
However,
the
routine
infant
formula
is
typically
produced
from
cow’s
milk,
where
the
␣-
lactalbumin
only
accounts
for
2%–5%.
␣-Lactalbumin
is
thus
often
added
as
a
strengthening
agent
in
the
infant
formula
to
match
the
nutrition
profile
in
breast
milk
[5,6].
This
creates
a
need
for
detecting
␣-lactalbumin
in
infant
formula
efficiently.
Liquid
chromatography–mass
spectrometry
(LC–MS)
has
become
the
main
method
for
the
determination
of
␣-lactalbumin
content,
due
to
its
advantages
of
high
sensitivity
and
high
selectiv-
∗
Corresponding
authors.
E-mail
addresses:
dachen@tju.edu.cn
(D.
Chen),
xgchu@vip.sina.com
(X.
Chu).
ity.
For
example,
Ren
and
Zhang
applied
high-performance
liquid
chromatography
(HPLC)-tandem
MS
with
␣-lactalbumin
signa-
ture
peptide
VGINYWLAHK,
isotope-labelled
signature
peptide
(ILSP)
VGI*NYWL*AHK,
whose
molar
conversion
quantitates
the
bovine
␣-lactalbumin
accurately.
Through
the
analysis
of
three
representative
tryptic
peptides
by
comparing
the
theoretical
and
endogenous
peptides,
VGINYWLAHK
was
selected
because
of
its
highest
abundance,
intensity
and
sensitivity
in
all
the
LC–MS/MS
analyses
[7,8].
However,
the
matrix
effect
may
distort
the
LC–MS
signals,
and
thus
spoiling
the
accuracy
and
precision
of
the
mea-
surement
[9,10].
In
order
to
validate
the
quantification
results
of
LC–MS,
the
US
Food
and
Drug
Administration
(FDA)
has
suggested
methods
to
reduce
the
matrix
effect
in
its
guidelines
in
developing
bioanalytical
methods
[11].
Among
the
current
methods
to
correct
matrix
effect,
the
internal
standard
method
is
the
most
effective
[12–14].
It
not
only
offsets
the
matrix
effect
of
ionization
[15,16],
but
also
eliminates
the
variations
in
the
sample
pretreatment
process
[17,18].
However,
the
usage
of
internal
standads
(IS)
could
https://doi.org/10.1016/j.chroma.2017.12.024
0021-9673/©
2017
Published
by
Elsevier
B.V.
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