Biology Reference
In-Depth Information
applications aimed at simultaneous monitoring from tens to
hundreds of MS/MS single-ion peptide transitions in a quantitative
manner. Ideally, these instruments are equipped with a high-
performance liquid chromatograph delivering LC eluent to the ESI
MS ion source with high accuracy at the micro- and nanofl ow rates.
The high accuracy of LC instruments allows the use of LC reten-
tion time as an important predictive and confi rmatory parameter
during the analysis of complex peptide mixtures. Triple stage qua-
drupole and hybrid quadrupole-ion trap instruments provide
unique surveillance scans such as precursor and product ion scans,
aiding in search for the fragmentation features that are common to
a family of compounds. In proteomics, this feature is often used to
fi nd posttranslationally modifi ed peptides. Commercial time-of-
fl ight (TOF) and hybrid (TOF/TOF) instruments predominate
MALDI-MS analyzers in the fi eld due to their high speed and rela-
tively lower costs. Many modern MS instruments provide high mass
resolution, extended mass range, high mass accuracy, good calibration
stability, and powerful “task-oriented” software.
There are numerous integrated MS data acquisition (operating
systems) and data processing software packages. Data acquisition soft-
ware operates an MS system and can make real-time choices during
the MS data acquisition. This level of automation is designed to opti-
mize data acquisition and results in minimum expenditure of time and
sample consumption. In contrast to the data acquisition software, data
processing software is usually installed on a computer or a computer
cluster separate from the one that controls the MS analytical system.
Modern proteomics software (ProteinPilot, GPS Explorer, Xcalibur,
Scaffold, ProteoIQ, MassLynx, etc.) provides appropriate tools for
protein identifi cation from the MS and MS/MS data using one or
more search engines (Mascot, Sequest, Peaks, Phenyx, X! Tandem,
etc.). DIGE images can be created and annotated with the MS protein
identifi cation and DIGE quantitation results. Systems Biology soft-
ware tools can then provide principal component and biological
pathway network analyses, to name but a few.
In this chapter, we discuss MALDI MS-based protein identifi -
cation from 2D DIGE-analyzed proteins using a 4800 MALDI-
TOF/TOF mass spectrometer. The techniques described in the
Subheadings
3
and
4
are routinely applied in our laboratory.
1.6. Software-Based
MS Data Analysis
and Verifi cation
of the Results
2. Materials
1.
Pipettors: 0.1-10, 2-200, and 1,000
μ
L.
2.1. General
Consumables
2.
Pipette tips: 0.1-10, 2-200, and 1,000
μ
L.
3.
0.6-mL microcentrifuge tubes (Axygen, MCT-060-L-C).
4.
1.5-mL microcentrifuge tubes (Axygen, MCT-150-L-C).
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