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Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Federale de Lausanne, 2 av. Forel, 1015 Lausanne, Switzerland
Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Leninskii Prospect 38, Bldg. 2,119334 Moscow,
Russia
Moscow Institute of Physics and Technology (State University), 9 Institutskiy per., 141707 Dolgoprudny, Moscow Region, Russia
S Supporting Information
*
(INTRODUCTION)
Bottom-up proteomics (BUP) is the current approach for highthroughput identication and quantitation of the proteins
present in a biological sample.1 This method entails digestion
of proteins into short (630 amino acid residue) peptides that
can be separated by liquid chromatography (LC) and analyzed
by tandem mass spectrometry (MS/MS). The robustness and
high throughput of the BUP approach combined with the stateof-the-art LCMS/MS technology allow identication and
quantitation of thousands of proteins with and without posttranslational modications in a single proteomic experiment.1,2
Modern high-resolution MS instruments, such as Orbitrap
Fourier transform mass spectrometer (FTMS), enable the
identication of up to 2500 proteins from a human sample in a
90 min LCMS/MS experiment.3 Signicant eorts on
optimization of the ionization and subsequent fragmentation
techniques have resulted in increased sensitivity and speed of MS
instruments in use.4 Despite these eorts, up to 85% of MS/MS
spectra acquired in a typical LC-MS/MS experiment remain
2013 American Chemical Society
Article
Article
Scheme 1. Classication of Mass Spectrometry-Based Proteomic Approaches Based on the Molecular Size of the Analytesa
(1) Type of LC column, (2) activation method, (3) mass analyzer, and (4) database search engine.
EXPERIMENTAL SECTION
In silico digestion of the Homo sapiens (human), Saccharomyces
cerevisiae (yeast), and Escherichia coli (bacteria) protein databases
(UniProt, release-2012_07) and calculation of peptide masses
were performed using the tools of in-house built open-source
Python library Pyteomics.30 We chose these three species as
representative data sets for the mammalian, fungal, and bacterial
kingdoms. The nonredundant databases contained 20 103
human, 6566 yeast, and 4243 bacteria proteins. Proteoforms
were not included in the calculations. In presenting the data, we
use the term unique peptides as accepted in the proteomics
literature. In brief, the term unique refers to peptides
representative of a single protein in a given proteome. In this
case, peptides that had a shared sequence between multiple
proteins were excluded from the statistics. For example, we
considered unique peptides for calculating the number of
proteins that could be potentially identied with a given pool
of peptides.
In the light of currently existing proteomic approaches, the
peptide size range identied by MDP and even the terminology
for the analysis of long peptides is not consistent. Fenselau and
others target the analysis of 310 kDa peptides and term the
analysis middle-down or middle-out proteomics,23,31 whereas
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Figure 1. Theoretical distribution of the total number of peptides and percentage of the proteins identiable with unique peptides in human (left
column), yeast (middle column), and bacteria (right column) proteomes after two-step consecutive cleavage with 7 kDa MW cuto after the rst
cleavage within (top row) 0.63, (middle row) 37, and (bottom row) 715 kDa ranges.
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suggesting that Kex2 digestion is most suitable when top-downtype analysis (>15 kDa peptide analysis) is sought, Figure 3,
bottom panel.
Aspartic Acid, Asp (D)
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2-(2-Nitrophenylsulfonyl)-3-methyl-3-bromoindolenine
(BNPS)-skatole is a brominating reagent that cleaves peptide
bonds C terminal to W.45 Another well-established protocol uses
o-iodosobenzoic acid; the yield is 80% with few side reactions. W
is one of the rarest amino acids in all three species studied;
however, the number of peptides in the BUP and eBUP regions
were comparable to those obtained in MDP ranges for all three
species (Figure 1). Yeast yielded 9270 BUP and 9529 eBUP
peptides (Figure 1b,e), whereas MDP and TDP ranges contained
8641 and 6535, respectively (Figure 1h,k). As expected, the
number of proteins identied by analysis of unique small and
midrange peptides was only 56.3 and 62.9%, respectively.
Unexpectedly, targeting W in the E. coli and human proteomes
resulted in a greater number of BUP peptides than in eBUP. This
could be indicating the preferential positioning of W residues
toward protein termini; cleavage at this residue resulting in a
short (630 amino acids) peptide and the complementary
remainder protein fragment. In E. coli, digestion at W residues
can enable identication of 66.6% and 70.0% of the proteins by
BUP and eBUP, respectively (as shown in the Venn diagram in
Figure S10, Supporting Information). A similar trend was
observed for the human database. Contrary to the expected
benet of MDP, combined BUP and eBUP analysis provided
unique peptides suitable for the identication of 85% of the
human proteins (Figure 5 top panel). This is a clear illustration of
how prior knowledge of the amino acid distribution of the
proteome can aid in decision on the working regime.
Article
Pairwise Cleavages
M followed by W
CNBr is a toxic reagent that has been used for decades for
attaining cleavages N-terminal to M.47 The three kingdoms
studied again presented dierent trends of peptide size
distribution when cleaved at M. Yeast showed comparable
number of peptides in the three mass ranges, allowing
identication of 80% of the proteome in either regime. A
dramatic dierence in the number of BUP peptides was obtained
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M followed by C
CONCLUSIONS
On the basis of our ndings, we propose that the analysis of
peptides in the 37 kDa range is more optimal than targeting
bottom-up (0.63 kDa) or middle-down (715 kDa) peptides
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Figure 7. Comparison of the performance of the BUP, eBUP, and MDP approaches for identication of the human proteome shown as a dependence on
the number of peptides on the percent of proteins represented by unique peptides. The total number of peptides shown on the y axis corresponds to
Figure 1 and describes peptides produced in a single digestion or in a two-step process with a cleavage at the rst amino acid, followed by a cleavage of
peptides >7 kDa at the second amino acid. Also shown are the data corresponding to the cleavages by trypsin and dibasic enzymes. (See Table S1 in the
Supporting Information.) The dotted black lines represent examples of detectable and identiable peptides in a 90 min and 4 h LCMS/MS
experiment, respectively. The dotted red line shows the total number of peptides detected from a 90 min LC gradient reported in the corresponding
reference.
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ASSOCIATED CONTENT
S Supporting Information
*
AUTHOR INFORMATION
Corresponding Author
*Phone: +41 21 693 97 51. Fax: +41 21 693 98 95. E-mail: yury.
tsybin@ep.ch.
Notes
ACKNOWLEDGMENTS
The work was supported by the Swiss National Science
Foundation (Projects 200021-125147 and 128357) and the
European Research Council (ERC Starting grant 280271 to
Y.O.T.). A.A.L. and M.V.G. thank EU FP7 Program support
(Project Prot-HiSPRA, grant agreement 282506).
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