4 research outputs found
Infrared Multiphoton Dissociation for Quantitative Shotgun Proteomics
We modified a dual-cell linear ion trap mass spectrometer
to perform
infrared multiphoton dissociation (IRMPD) in the low-pressure trap
of a dual-cell quadrupole linear ion trap (dual-cell QLT) and perform
large-scale IRMPD analyses of complex peptide mixtures. Upon optimization
of activation parameters (precursor <i>q</i>-value, irradiation
time, and photon flux), IRMPD subtly, but significantly, outperforms
resonant-excitation collisional-activated dissociation (CAD) for peptides
identified at a 1% false-discovery rate (FDR) from a yeast tryptic
digest (95% confidence, <i>p</i> = 0.019). We further demonstrate
that IRMPD is compatible with the analysis of isobaric-tagged peptides.
Using fixed QLT rf amplitude allows for the consistent retention of
reporter ions, but necessitates the use of variable IRMPD irradiation
times, dependent upon precursor mass to charge (<i>m</i>/<i>z</i>). We show that IRMPD activation parameters can
be tuned to allow for effective peptide identification and quantitation
simultaneously. We thus conclude that IRMPD performed in a dual-cell
ion trap is an effective option for the large-scale analysis of both
unmodified and isobaric-tagged peptides
Accurate Multiplexed Proteomics at the MS2 Level Using the Complement Reporter Ion Cluster
Isobaric labeling strategies, such as isobaric tags for
relative
and absolute quantitation (iTRAQ) or tandem mass tags (TMT), have
promised to dramatically increase the power of quantitative proteomics.
However, when applied to complex mixtures, both the accuracy and precision
are undermined by interfering peptide ions that coisolate and cofragment
with the target peptide. Additional gas-phase isolation steps, such
as proton-transfer ion–ion reactions (PTR) or higher-order
MS3 scans, can almost completely eliminate this problem. Unfortunately,
these methods come at the expense of decreased acquisition speed and
sensitivity. Here we present a method that allows accurate quantification
of TMT-labeled peptides at the MS2 level without additional ion purification.
Quantification is based on the fragment ion cluster that carries most
of the TMT mass balance. In contrast to the use of low <i>m</i>/<i>z</i> reporter ions, the localization of these complement
TMT (TMT<sup>C</sup>) ions in the spectrum is precursor-specific;
coeluting peptides do not generally affect the measurement of the
TMT<sup>C</sup> ion cluster of interest. Unlike the PTR or MS3 strategies,
this method can be implemented on a wide range of high-resolution
mass spectrometers like the quadrupole Orbitrap instruments (QExactive).
A current limitation of the method is that the efficiency of TMT<sup>C</sup> ion formation is affected by both peptide sequence and peptide
ion charge state; we discuss potential routes to overcome this problem.
Finally, we show that the complement reporter ion approach allows
parallelization of multiplexed quantification and therefore holds
the potential to multiply the number of distinct peptides that can
be quantified in a given time frame
Comprehensive Single-Shot Proteomics with FAIMS on a Hybrid Orbitrap Mass Spectrometer
Liquid
chromatography (LC) prefractionation is often implemented
to increase proteomic coverage; however, while effective, this approach
is laborious, requires considerable sample amount, and can be cumbersome.
We describe how interfacing a recently described high-field asymmetric
waveform ion mobility spectrometry (FAIMS) device between a nanoelectrospray
ionization (nanoESI) emitter and an Orbitrap hybrid mass spectrometer
(MS) enables the collection of single-shot proteomic data with comparable
depth to that of conventional two-dimensional LC approaches. This
next generation FAIMS device incorporates improved ion sampling at
the ESI–FAIMS interface, increased electric field strength,
and a helium-free ion transport gas. With fast internal compensation
voltage (CV) stepping (25 ms/transition), multiple unique gas-phase
fractions may be analyzed simultaneously over the course of an MS
analysis. We have comprehensively demonstrated how this device performs
for bottom-up proteomics experiments as well as characterized the
effects of peptide charge state, mass loading, analysis time, and
additional variables. We also offer recommendations for the number
of CVs and which CVs to use for different lengths of experiments.
Internal CV stepping experiments increase protein identifications
from a single-shot experiment to >8000, from over 100 000
peptide
identifications in as little as 5 h. In single-shot 4 h label-free
quantitation (LFQ) experiments of a human cell line, we quantified
7818 proteins with FAIMS using intra-analysis CV switching compared
to 6809 without FAIMS. Single-shot FAIMS results also compare favorably
with LC fractionation experiments. A 6 h single-shot FAIMS experiment
generates 8007 protein identifications, while four fractions analyzed
for 1.5 h each produce 7776 protein identifications
Improved Precursor Characterization for Data-Dependent Mass Spectrometry
Modern
ion trap mass spectrometers are capable of collecting up
to 60 tandem MS (MS/MS) scans per second, in theory providing acquisition
speeds that can sample every eluting peptide precursor presented to
the MS system. In practice, however, the precursor sampling capacity
enabled by these ultrafast acquisition rates is often underutilized
due to a host of reasons (e.g., long injection times and wide analyzer
mass ranges). One often overlooked reason for this underutilization
is that the instrument exhausts all the peptide features it identifies
as suitable for MS/MS fragmentation. Highly abundant features can
prevent annotation of lower abundance precursor ions that occupy similar
mass-to-charge (<i>m</i>/<i>z</i>) space, which
ultimately inhibits the acquisition of an MS/MS event. Here, we present
an advanced peak determination (APD) algorithm that uses an iterative
approach to annotate densely populated <i>m</i>/<i>z</i> regions to increase the number of peptides sampled during
data-dependent LC-MS/MS analyses. The APD algorithm enables nearly
full utilization of the sampling capacity of a quadrupole-Orbitrap-linear
ion trap MS system, which yields up to a 40% increase in unique peptide
identifications from whole cell HeLa lysates (approximately 53 000
in a 90 min LC-MS/MS analysis). The APD algorithm maintains improved
peptide and protein identifications across several modes of proteomic
data acquisition, including varying gradient lengths, different degrees
of prefractionation, peptides derived from multiple proteases, and
phosphoproteomic analyses. Additionally, the use of APD increases
the number of peptides characterized per protein, providing improved
protein quantification. In all, the APD algorithm increases the number
of detectable peptide features, which maximizes utilization of the
high MS/MS capacities and significantly improves sampling depth and
identifications in proteomic experiments