Biology Reference
In-Depth Information
chosen peptides are synthesized, including (at least) one stable
isotope labeled amino acid (being highly enriched with
15
N and
13
C) per peptide. In contrast to the latter, within the experimental
approach, proteotypic peptides are chosen through data mining of
shotgun-proteomics experiments, mass spectral reference data-
bases such as the plant proteomic spectral library ProMEX [
4
], as
well as protein fractionation, enrichment, and subsequent mass
spectral analysis. The ensuing synthesis of stable isotope labeled
peptides is identical to the theoretical approach, as well as the rest
of the procedure. LC-SRM/MS method development follows
and completes the workfl ow of the Mass Western approach. The
detailed workfl ow is presented in the specifi c context of quantifi -
cation capacity of a QqQ, here a TSQ (Thermo Triple Stage
Quadrupole Vantage), compared to a Linear Trap Quadrupole-
Orbitrap (LTQ-Orbitrap).
2
Materials
We recommend the use of Skyline ([
5
],
https://brendanx-uw1.
gs.washington.edu/
)
for method development and data analysis.
Proteolytic cleavage probability, hydrophobicity calculation, and
PTM prediction are part of many useful features available at Expasy
Tools (
http://ca.expasy.org/
). Skyline in conjunction with SRM
collider can be used for the predition of proteotypic peptides and
to fi nd interferences in a given background proteome [
6
].
2.1 Software
TSQ (Thermo Triple Stage Quadrupole, Vantage).
LTQ-Orbitrap (Thermo Linear Trap Quadrupole-Orbitrap XL).
2.2 Mass
Spectrometer
3
Methods
3.1 General
Overview
The heavy synthetic standard peptide (containing one amino acid
labeled with
15
N and
13
C; HP) and its native counterpart, the light
peptide (with no artifi cially introduced label; LP), share identical
physicochemical properties. Retention time, ionization effi ciency,
and fragmentation properties of a HP-LP pair are assumed to be
identical. The HP is used to tune the mass spectrometer and to
create an external calibration curve. The peak area of the LP is set
into the calibration curve to calculate the absolute quantity. When
measuring samples for quantifi cation, equal amounts of HP are
spiked into each sample, as internal standards. The internal stan-
dard serves as a quality control. The retention time, peak shape,
and relative intensities of individual transitions (
see
Notes 1
and
2
)
of the LP are compared to the HP. This gives the experimenter
great confi dence concerning the accuracy of the data used for
