Improved phosphoproteome analysis
by multi-enzymatic digestions targeting the Trypsin-Resistant Proteome (TReP)
Bao Tran1; Celine Hernandez1, 2; Alexandra Potts1; Patrice Waridel1; Frederique Lisacek2; Manfredo Quadroni1
1 University of Lausanne, Lausanne, Switzerland; 2 Swiss Institute of Bioinformatics, Geneva, Switzerland
OVERVIEW
2. METHOD
3.3. Improvement of LC-MS/MS mapping of
large tryptic peptides
3. RESULTS
NL: 2.50E7
Total number of AA
4.00E+05
Isolation of large peptides by SEC and digestion with
four cleaving agents yields MS-suitable products, with
increase of sequence coverage and identification:
Small peptide
3.00E+05
Medium peptide
Higher peptide
2.00E+05
All peptides
1.00E+05
Application to human phosphoproteomics : 410
unique (not found with trypsin alone) phosphosites, of
which 41% are not annotated in databases
0.00E+00
1
11
21
31
41
51
61
71
81
91
Length of peptide (AA)
Proteome covered by:
Comparison: only 8% non-annotated phosphosites in
our trypsin data
“small” peptides (≥ 800 Da): 19.3%
TReP analysis reveals a «hidden» part of the proteome
“large” peptides (≥3000Da): 25.0%
“medium” peptides (3000>Mw>800Da): 55.7%
80
70
60
50
40
30
20
60
40
20
10
0
30
40
50
Time (min)
60
70
400 600 800 10001200140016001800
m/z
30
0
20
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
50
10
10
Trypsin+
Glu-C
Relative Abundance
Trypsin
Relative Abundance
Covered sequence as
function of
peptide
length
(1’218’741 peptides of
human
proteome,
Uniprot/Swissprot
version 15.13),
full trypsin cleavage
SDNEDNDFDEDDEDDAALVAAMTNAPGNSLDIEESVGYGATSAPTSNTNHVVESANAAYYQR
90
80
Relative Abundance
In silico tryptic peptides of human proteome
5.00E+05
100
90
70
Identification of large peptides by shotgun MS is not
efficient
NL: 2.50E7
100
3.1. In silico tryptic peptide analysis
20-30% of a trypsinised proteome are constituted of
peptides with Mw≥3000 (TReP)
80
0
10
20
30
40
50
Time (min)
60
70
80
Total Ion Current (TIC) of SEC fraction 3 of sample SCVMprot
analyzed directly and after Glu-C digestion.
The inset spectrum was acquired at RT=31.21 min and assigned to
one peptide (displayed in red) of protein ID B3LQ90_YEAS1. The
corresponding extended tryptic peptide shown is 63 AA long (Mw:
6.5 kDa).
Second digestion may lead to the generation of analyzable
fragments out of much larger peptides which, in their intact
form, would be very difficult to detect and identify by LCMS/MS.
3.4. Phosphopeptide analysis
Concept : after trypsin digestion, isolation of all polypeptides
with Mw ≥ 3000 Da, and digestion with alternative proteases
Amino acid frequency ratio
of in silico large tryptic
peptides
(69’571)
to
medium ones (465’160) in
human proteome
Amino acid frequency ratio
of large human peptides vs medium peptides
Analysis of complex samples by liquid chromatographyelectrospray MS after trypsin cleavage is the most popular
approach
for
bottom-up
proteomics.
However,
comprehensive mapping of post-translational modifications
is limited, among other things, by the presence of long
sequences without trypsin cleavage sites.
We tried to:
Complement the trypsin-based workflow with a strategy
that specifically targets the fraction of the proteome that
cannot be digested by trypsin (the Trypsin-Resistant
Proteome, TReP), in order to give compatible masses for
shotgun MS
Saccharomyces cerevisiae vacuolar membrane fraction
(SCVMprot)
Human melanoma cell line lysate (SKMel28 cells)
1.2
1
0.8
0.6
0.4
0.2
SEC fractionation: 11 fractions on Äkta purifier 10 system,
Superdex Peptide 10/300 GL column
RPLC-MS/MS analysis: Agilent nano 1100 HPLC system
coupled to LTQ-Orbitrap-XL
Data processing
0
K
R
E
D
N
H
Q
Y
L
W
C
V
A
G
F
T
S
P
Amino acid
MS/MS spectra sets of
trypsin or 2nd enzymes
Uniprot
Phospho-database
Phosphosite.org
Phospho-database
K.QPPPNMIFNPNQNPMANQEQQNQSIFHQQSNMAPMNQEQQPMQFQ
SQSTVSSLQNPGPTQSESSQTPLFHSSPQIQLVQGSPSSQEQQVTLFLS
PASMSALQTSINQQDMQQSPLYSPQNNMPGIQGATSSPQPQATLFHNT
AGGTMNQLQNSPGSSQQTSGMFLFGIQNNCSQLLTSGPATLPDQLMAI
SQPGQPQNEGQPPVTTLLSQQMPENpSPLASSINTNQNIEK.I
The phosphosite (pS) and the protein phosphorylation were
not annotated.
3.2. Fractionation of tryptic digests according
to peptide size
Peptide Mw ≥ 2.4 kDa
Peptide Mw < 2.4 kDa
LogMw
5
4.5
31kDa
4
10kDa
800
3.5
3.1kDa
3
1.0kDa
2.5
0.3kDa
Composition of SKMel phosphosites
No
New
phosphoproteins
Phosphoprotein ?
400
800
928
743
trypsin
688
600
400
240
Enhance sequence coverage
1.5
200
No
1
Site Annotated ?
Increase number of phosphosite identifications.
0.5
0
Phosphopeptides
Ascore ≥ 19
Yes
1 2 3 4 5 6 7 8 9 1011
0.0
Annotated phosphosites
New phosphosites
5.0
55
84
43
72 66
chymo
Glu-C
66 56
54
Asp-N
FA
Phosphorylation sites:
Localization: Ascore ≥ 19 ref 1
Site mapping (custom-made scripts):
• Compare the phosphosite sets from different proteases
• Find the new sites not annotated by Uniprot and
phosphosite.org databases
15.0
20.0
25.0 ml
4 second
enzymes
Total
Protease
non-annotated sites
Identified:
• SCVMprot: 80
µg
• SKMel: 4 mg
• Collection of 11
fractions of 1mL
with
410 other phosphosites
with 4 second enzymes
17
0
trypsin
phosphosites
225 sites not annotated in
Uniprot/Swiss-prot
and
phophosite.org databases
703 phosphorylated peptides; 440 phosphoproteins (19
not annotated)
4. CONCLUSION
Calibration curve: logMw = -0.2088V + 5.9066
____ SCVMprot
PHOSPHOSITE MAPPING
10.0
225
170
200
annotated sites
2
Yes
Localized:
1000
600
Database search
Ascore
1. Beausoleil, S. A., Villen, J., Gerber, S. A., Rush, J.,
and Gygi, S. P. (2006) A probability-based approach for
high-throughput protein phosphorylation analysis and
site localization. Nat Biotech 24, 1285-1292.
I
mAU
Apply the method to characterization of trypsinresistant protein sequences:
5. REFERENCE
M
Frequency
of
S,T,Y
residues in large peptides
is higher than in medium
peptides.
Example: Tryptic phosphopeptide (24.8 kDa) of protein
NFAT5_HUMAN, and the corresponding peptide generated
with Glu-C (red, identified in fraction 3).
Number of phospho sites
Samples:
1. INTRODUCTION
Amino acid frequency ratio
1.4
____ Human SKMel
Fractions 1-6 containing large peptide were subjected to
2nd digestion with other cleaving agents.
Fraction 3 of SCVMprot was used to examine LC-MS
behavior of large tryptic peptides before and after second
digestion (Glu-C).
Fractions of SKMel
identification experiments.
were
used
for
phosphosite
The developed method uses a second digestion step
targeting large tryptic peptides and was successfully employed
to discover trypsin-inaccessible phosphorylation sites in the
proteome of a human melanoma cell line, significantly
enhancing the number of phosphosites.
Many of the identified phosphosites and phosphoproteins
have not been found in UNIPROT and phosphosite.org
databases.
This strategy complements regular trypsin-based analysis.