15 research outputs found
Distinguishing Analyte from Noise Components in Mass Spectra of Complex Samples: Where to Cut the Noise?
Fourier
transform mass spectrometry (FTMS) enables comprehensive
analysis of complex molecular mixtures. Given the broad intensity
ranges of components in the mass spectra, it is imperative to accurately
determine a noise threshold level above which peak assignments will
be made. Conventionally, to find the threshold level, the â<i>N</i> sigmaâ approach or an equivalent rule is used.
However, the â<i>N</i> sigmaâ approach cannot
be applied to mass spectra stored with partially removed noise (reduced-profile
mode). It is also not directly applicable to mass spectra acquired
in the absorption mode with removed negative spectral amplitudes.
Moreover, <i>N</i> value selection is normally made based
on a rule of thumb, meaning that the calculated threshold level may
be biased. Here, we present a noise thresholding method which addresses
these limitations for analysis of mass spectra of complex molecular
mixtures. The introduced data-dependent thresholding method involves
analysis of the distribution of logarithmic intensity of all peaks,
including noise and analyte, for a given mass spectrum. Selected method
applications include FTMS analysis of crude oil fractions as well
as tandem MS analysis of intact proteins
Middle-Down Analysis of Monoclonal Antibodies with Electron Transfer Dissociation Orbitrap Fourier Transform Mass Spectrometry
The rapid growth of approved biotherapeutics,
e.g., monoclonal
antibodies or immunoglobulins G (IgGs), demands improved techniques
for their quality control. Traditionally, proteolysis-based bottom-up
mass spectrometry (MS) has been employed. However, the long, multistep
sample preparation protocols required for bottom-up MS are known to
potentially introduce artifacts in the original sample. For this reason,
a top-down MS approach would be preferable. The current performance
of top-down MS of intact monoclonal IgGs, though, enables reaching
only up to âŒ30% sequence coverage, with incomplete sequencing
of the complementarity determining regions which are fundamental for
IgGâs antigen binding. Here, we describe a middle-down MS protocol
based on the use of immunoglobulin G-degrading enzyme of <i>Streptococcus
pyogenes</i> (IdeS), which is capable of digesting IgGs in only
30 min. After chemical reduction, the obtained âŒ25 kDa proteolytic
fragments were analyzed by reversed phase liquid chromatography (LC)
coupled online with an electron transfer dissociation (ETD)-enabled
hybrid Orbitrap Fourier transform mass spectrometer (Orbitrap Elite
FTMS). Upon optimization of ETD and product ion transfer parameters,
results show that up to âŒ50% sequence coverage for selected
IgG fragments is reached in a single LC run and up to âŒ70%
when data obtained by distinct LCâMS runs are averaged. Importantly,
we demonstrate the potential of this middle-down approach in the identification
of oxidized methionine residues. The described approach shows a particular
potential for the analysis of IgG mixtures
Quantitation and Identification of Thousands of Human Proteoforms below 30 kDa
Top-down proteomics is capable of
identifying and quantitating
unique proteoforms through the analysis of intact proteins. We extended
the coverage of the label-free technique, achieving differential analysis
of whole proteins <30 kDa from the proteomes of growing and senescent
human fibroblasts. By integrating improved control software with more
instrument time allocated for quantitation of intact ions, we were
able to collect protein data between the two cell states, confidently
comparing 1577 proteoform levels. To then identify and characterize
proteoforms, our advanced acquisition software, named Autopilot, employed enhanced identification efficiency in identifying 1180
unique Swiss-Prot accession numbers at 1% false-discovery rate. This
coverage of the low mass proteome is equivalent to the largest previously
reported but was accomplished in 23% of the total acquisition time.
By maximizing both the number of quantified proteoforms and their
identification rate in an integrated software environment, this work
significantly advances proteoform-resolved analyses of complex systems
Quantitation and Identification of Thousands of Human Proteoforms below 30 kDa
Top-down proteomics is capable of
identifying and quantitating
unique proteoforms through the analysis of intact proteins. We extended
the coverage of the label-free technique, achieving differential analysis
of whole proteins <30 kDa from the proteomes of growing and senescent
human fibroblasts. By integrating improved control software with more
instrument time allocated for quantitation of intact ions, we were
able to collect protein data between the two cell states, confidently
comparing 1577 proteoform levels. To then identify and characterize
proteoforms, our advanced acquisition software, named Autopilot, employed enhanced identification efficiency in identifying 1180
unique Swiss-Prot accession numbers at 1% false-discovery rate. This
coverage of the low mass proteome is equivalent to the largest previously
reported but was accomplished in 23% of the total acquisition time.
By maximizing both the number of quantified proteoforms and their
identification rate in an integrated software environment, this work
significantly advances proteoform-resolved analyses of complex systems
Quantitation and Identification of Thousands of Human Proteoforms below 30 kDa
Top-down proteomics is capable of
identifying and quantitating
unique proteoforms through the analysis of intact proteins. We extended
the coverage of the label-free technique, achieving differential analysis
of whole proteins <30 kDa from the proteomes of growing and senescent
human fibroblasts. By integrating improved control software with more
instrument time allocated for quantitation of intact ions, we were
able to collect protein data between the two cell states, confidently
comparing 1577 proteoform levels. To then identify and characterize
proteoforms, our advanced acquisition software, named Autopilot, employed enhanced identification efficiency in identifying 1180
unique Swiss-Prot accession numbers at 1% false-discovery rate. This
coverage of the low mass proteome is equivalent to the largest previously
reported but was accomplished in 23% of the total acquisition time.
By maximizing both the number of quantified proteoforms and their
identification rate in an integrated software environment, this work
significantly advances proteoform-resolved analyses of complex systems
In-Spray Supercharging of Peptides and Proteins in Electrospray Ionization Mass Spectrometry
Enhanced charging, or supercharging, of analytes in electrospray
ionization mass spectrometry (ESI MS) facilitates high resolution
MS by reducing an ion mass-to-charge (<i>m</i>/<i>z</i>) ratio, increasing tandem mass spectrometry (MS/MS) efficiency.
ESI MS supercharging is usually achieved by adding a supercharging
reagent to the electrospray solution. Addition of these supercharging
reagents to the mobile phase in liquid chromatography (LC)-MS/MS increases
the average charge of enzymatically derived peptides and improves
peptide and protein identification in large-scale bottom-up proteomics
applications but disrupts chromatographic separation. Here, we demonstrate
the average charge state of selected peptides and proteins increases
by introducing the supercharging reagents directly into the ESI Taylor
cone (in-spray supercharging) using a dual-sprayer ESI microchip.
The results are comparable to those obtained by the addition of supercharging
reagents directly into the analyte solution or LC mobile phase. Therefore,
supercharging reaction can be accomplished on a time-scale of ion
liberation from a droplet in the ESI ion source
Advantages of Extended Bottom-Up Proteomics Using Sap9 for Analysis of Monoclonal Antibodies
Despite the recent advances in structural
analysis of monoclonal
antibodies with bottom-up, middle-down, and top-down mass spectrometry
(MS), further improvements in analysis accuracy, depth, and speed
are needed. The remaining challenges include quantitatively accurate
assignment of post-translational modifications, reduction of artifacts
introduced during sample preparation, increased sequence coverage
per liquid chromatography (LC) MS experiment, and ability to extend
the detailed characterization to simple antibody cocktails and more
complex antibody mixtures. Here, we evaluate the recently introduced
extended bottom-up proteomics (eBUP) approach based on proteolysis
with secreted aspartic protease 9, Sap9, for analysis of monoclonal
antibodies. Key findings of the Sap9-based proteomics analysis of
a single antibody include: (i) extensive antibody sequence coverage
with up to 100% for the light chain and up to 99â100% for the
heavy chain in a single LC-MS run; (ii) connectivity of complementarity-determining
regions (CDRs) via Sap9-produced large proteolytic peptides (3.4 kDa
on average) containing up to two CDRs per peptide; (iii) reduced artifact
introduction (e. g., deamidation) during proteolysis with Sap9 compared
to conventional bottom-up proteomics workflows. The analysis of a
mixture of six antibodies via Sap9-based eBUP produced comparable
results. Due to the reasons specified above, Sap9-produced proteolytic
peptides improve the identification confidence of antibodies from
the mixtures compared to conventional bottom-up proteomics dealing
with shorter proteolytic peptides
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Chemical-Mediated Digestion: An Alternative Realm for Middle-down Proteomics?
Protein digestion in mass spectrometry
(MS)-based bottom-up proteomics
targets mainly lysine and arginine residues, yielding primarily 0.6â3
kDa peptides for the proteomes of organisms of all major kingdoms.
Recent advances in MS technology enable analysis of complex mixtures
of increasingly longer (>3 kDa) peptides in a high-throughput manner
supporting the development of a middle-down proteomics (MDP) approach.
Generating longer peptides is a paramount step in launching an MDP
pipeline, but the quest for the selection of a cleaving agent that
would provide the desired 3â15 kDa peptides remains open. Recent
bioinformatics studies have shown that cleavage at the rarely occurring
amino acid residues such as methionine (Met), tryptophan (Trp), or
cysteine (Cys) would be suitable for MDP approach. Interestingly,
chemical-mediated proteolytic cleavages uniquely allow targeting these
rare amino acids, for which no specific proteolytic enzymes are known.
Herein, as potential candidates for MDP-grade proteolysis, we have
investigated the performance of chemical agents previously reported
to target primarily Met, Trp, and Cys residues: CNBr, BNPS-Skatole
(3-bromo-3-methyl-2-(2-nitrophenyl)Âsulfanylindole), and NTCB
(2-nitro-5-thiobenzoic acid), respectively. Figures of merit such
as digestion reproducibility, peptide size distribution, and occurrence
of side reactions are discussed. The NTCB-based MDP workflow has demonstrated
particularly attractive performance, and NTCB is put forward here
as a potential cleaving agent for further MDP development
Chemical-Mediated Digestion: An Alternative Realm for Middle-down Proteomics?
Protein digestion in mass spectrometry
(MS)-based bottom-up proteomics
targets mainly lysine and arginine residues, yielding primarily 0.6â3
kDa peptides for the proteomes of organisms of all major kingdoms.
Recent advances in MS technology enable analysis of complex mixtures
of increasingly longer (>3 kDa) peptides in a high-throughput manner
supporting the development of a middle-down proteomics (MDP) approach.
Generating longer peptides is a paramount step in launching an MDP
pipeline, but the quest for the selection of a cleaving agent that
would provide the desired 3â15 kDa peptides remains open. Recent
bioinformatics studies have shown that cleavage at the rarely occurring
amino acid residues such as methionine (Met), tryptophan (Trp), or
cysteine (Cys) would be suitable for MDP approach. Interestingly,
chemical-mediated proteolytic cleavages uniquely allow targeting these
rare amino acids, for which no specific proteolytic enzymes are known.
Herein, as potential candidates for MDP-grade proteolysis, we have
investigated the performance of chemical agents previously reported
to target primarily Met, Trp, and Cys residues: CNBr, BNPS-Skatole
(3-bromo-3-methyl-2-(2-nitrophenyl)Âsulfanylindole), and NTCB
(2-nitro-5-thiobenzoic acid), respectively. Figures of merit such
as digestion reproducibility, peptide size distribution, and occurrence
of side reactions are discussed. The NTCB-based MDP workflow has demonstrated
particularly attractive performance, and NTCB is put forward here
as a potential cleaving agent for further MDP development