3 research outputs found
Multiple and Sequential Data Acquisition Method: An Improved Method for Fragmentation and Detection of Cross-Linked Peptides on a Hybrid Linear Trap Quadrupole Orbitrap Velos Mass Spectrometer
The identification and validation of cross-linked peptides
by mass
spectrometry remains a daunting challenge for proteināprotein
cross-linking approaches when investigating protein interactions.
This includes the fragmentation of cross-linked peptides in the mass
spectrometer per se and following database searching, the matching
of the molecular masses of the fragment ions to the correct cross-linked
peptides. The hybrid linear trap quadrupole (LTQ) Orbitrap Velos combines
the speed of the tandem mass spectrometry (MS/MS) duty circle with
high mass accuracy, and these features were utilized in the current
study to substantially improve the confidence in the identification
of cross-linked peptides. An MS/MS method termed multiple and sequential
data acquisition method (MSDAM) was developed. Preliminary optimization
of the MS/MS settings was performed with a synthetic peptide (TP1)
cross-linked with bisĀ[sulfosuccinimidyl] suberate (BS<sup>3</sup>).
On the basis of these results, MSDAM was created and assessed on the
BS<sup>3</sup>-cross-linked bovine serum albumin (BSA) homodimer.
MSDAM applies a series of multiple sequential fragmentation events
with a range of different normalized collision energies (NCE) to the
same precursor ion. The combination of a series of NCE enabled a considerable
improvement in the quality of the fragmentation spectra for cross-linked
peptides, and ultimately aided in the identification of the sequences
of the cross-linked peptides. Concurrently, MSDAM provides confirmatory
evidence from the formation of reporter ions fragments, which reduces
the false positive rate of incorrectly assigned cross-linked peptides
Combining Filter-Aided Sample Preparation and Pseudoshotgun Technology To Profile the Proteome of a Low Number of Early Passage Human Melanoma Cells
The performance of two proteomic sample preparation methods,
āpseudoshotgunā
(PSG) and filter-aided sample preparation (FASP) were compared in
terms of the number of identified proteins, representation of cellular
component GO (gene ontology) categories in the obtained list of proteins,
and the efficiency of both methods in the proteomic analysis of a
very low number of cells. Both methods were combined to obtain a proteomic
profile of a short-term culture (passage 3) of melanoma cells, established
in our laboratory from a human metastatic melanoma lesion. The data
revealed that with FASP, usually more proteins are identified than
with PSG when analyzing a higher number of cells (ā„5000/injection),
whereas PSG is favorable when analyzing only a very small amount of
cells (250ā500/injection). PSG and FASP, however, are complementary
techniques, as combining both methods further increases the number
of identified proteins. Moreover, we show that it is feasible to identify
a substantial number of proteins from only 250 cells/injection that
is equivalent to 60 ng of protein
Combining Filter-Aided Sample Preparation and Pseudoshotgun Technology To Profile the Proteome of a Low Number of Early Passage Human Melanoma Cells
The performance of two proteomic sample preparation methods,
āpseudoshotgunā
(PSG) and filter-aided sample preparation (FASP) were compared in
terms of the number of identified proteins, representation of cellular
component GO (gene ontology) categories in the obtained list of proteins,
and the efficiency of both methods in the proteomic analysis of a
very low number of cells. Both methods were combined to obtain a proteomic
profile of a short-term culture (passage 3) of melanoma cells, established
in our laboratory from a human metastatic melanoma lesion. The data
revealed that with FASP, usually more proteins are identified than
with PSG when analyzing a higher number of cells (ā„5000/injection),
whereas PSG is favorable when analyzing only a very small amount of
cells (250ā500/injection). PSG and FASP, however, are complementary
techniques, as combining both methods further increases the number
of identified proteins. Moreover, we show that it is feasible to identify
a substantial number of proteins from only 250 cells/injection that
is equivalent to 60 ng of protein