Biology Reference
In-Depth Information
accurate as with external calibration curves. An external calibration
curve is recorded as follows. A dilution series of the synthetic pep-
tides is measured using optimized settings for the instrument given.
The linear dynamic range (LDR), the limit of detection (LOD), and
the limit of quantifi cation (LOQ) can thereby be estimated. If the
standard peptides are spiked into a complex matrix (refl ecting the
nature of the sample to be analyzed), the LDR, LOD, and LOQ in
matrix can be assessed more precisely concerning the performance
of the assay. Accurate quantifi cation can only be achieved within the
linear dynamic range. For each HP a linear regression is calculated
for data within its linear dynamic range.
3.7 Absolute
Quantifi cation
When measuring samples for quantifi cation, the amount of HP
added to the individual samples should be kept constant. The
heavy standard peptide (HP) is introduced to the sample at the
earliest possible point of time. The signal intensity of the HP stan-
dard should be as close as possible to that of the native target. The
peak area integral of the HP can be used to normalize data across
multiple samples. The LP peak area can thus be adjusted and set
into the regression equation of the external calibration curve to
calculate a quantitative value. Best results are expected if the values
are in the middle of the linear regression.
3.8 Frequently
Asked Question: Triple
Quadrupole or Orbitrap
The SRM approach can generally be executed on different types of
MS. At present, for absolute quantifi cation triple quadrupole mass
spectrometers (QqQ) are routinely being used due to a wide linear
dynamic range, excellent sensitivity and selectivity, as well as acqui-
sition speed. In contrast, due to the very long duty cycles and
incomparable sensitivity, quantifi cation based on SRM on the
hybrid MS instrument LTQ-Orbitrap-XL is not recommended.
The LTQ-Orbitrap-XL MS features high mass accuracy and
resolution, and is an excellent tool for discovery experiments (such
as shotgun proteomics), and relative quantifi cation [
7
,
8
]. The lat-
ter MS instrument has also successfully been used for LC-MS based
quantifi cation with the drawback of a reduced linear dynamic range
compared to QqQ LC-SRM/MS. Recent articles state that the
Orbitrap-Exactive is “equal or better” than QqQ if full scan acquisi-
tion based quantifi cation is applied [
9
,
10
] (
see
Note 3
). However,
at present the use of QqQ holds advantages compared to other
types of instruments such as a Linear Trap Quadrupole-Orbitrap
(LTQ-Orbitrap). Figure
3
illustrates the differing linear dynamic
range of the aforementioned instruments for three distinct standard
HP. The two MS instruments are compared for applicability of
absolute quantifi cation (methodological details can be found at
http://www.univie.ac.at/mosys/publications.html
)
. The fi gure
shows that even though LTQ-Orbitrap MS signal intensities in data
dependent mode appears higher, the linear dynamic range is bigger
for the QqQ Triple Stage Quadrupole (TSQ) MS (
see
Note 4
).
