processing of Skyline MS1 Filtering data sets on a Skyline workstation:

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7 Νοε 2013 (πριν από 4 χρόνια και 1 μήνα)

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Installing the “MS1Probe” tool and corresponding Python script for further statistical
processing of Skyline MS1 Filtering data sets on a Skyline workstation:


Python is an object oriented programming language to organize and analyze data. The current
release of Python, version 2.7.3,
should

be downloaded at
http://www.python.org/download/
.

Note: Python
v.
3.3 may
c
ause compatibility problems
,

currently
MS1Probe requires
P
ython
v2.7.3.

(for further details

and final setup of MS1Probe

see below).



A.
Description of MS1 Probe Functionalities

and statistical processing capabilities
:



The Python program

referred to as ‘MS1Probe’ will be implemented as
a
Skyline Tool into
Skyline itself using ‘SkylineRunner’ as described in detail below. Upon starting of the
MS1Probe Tool within Skyline, several automatized steps will take place
, overview
:

i)

Skyline will g
enerate a custom results report in comma delimited format (*.csv file)
from Skyline MS1 Filtering data;

ii)

T
he

‘MS1Probe’

program
will
prompt the user
in a command window
to enter the
number of
different
conditions

and/or sample types (i.e., cancer/control o
r
wildtype/mutant strain etc
.
)

in
the

data

sets

and the

number
and types
of statistical

ratios

(between the different conditions)

that
should be calculated
;

iii)

‘MS1Probe’

extracts the data from
the automatically generated Skyline results report
,
and
populates a data
array
for all MS1 precursor ions segregat
ing replicates

by

custom conditions that the user had defined in the above steps (i.e. cancer/control
type samples)

iv)

Precursor

P
eak
A
rea
M
ean
s

are

calculated between the replicates of the specifie
d
condition, along with the
s
tandard
d
eviation and the
c
oefficient of
v
ariation.

R
atios
are calculated using the mean of the peak areas
from all replicates
per condition
(per
sample type, i.e., WT and KO, respectively)
along with
p
-
values

to assess
signif
icance

(Student’s
two
-
tailed

t
-
test
).

v)

MS1Probe

will prompt the user to append
a name

annotation to the
generated
Skyline
report and

the
final

Statistic
s

Output.

The
two
output files (
*
.csv) are generated and
placed in
to

the

utilized Skyline File

(
*
.sky)
source

folder.


The Statistics

Output

file
will automatically
be opened in
E
xcel
.

B.
Preparations and
Installations

I
nstallation and setup of the Skyline external tool ‘MS1Probe’ for automated generation of
statistical reports for MS1 Filtering
Quantitation (
i
nstructions for Windows
computers

only
)
.

1.)

Install
ation of

the latest version of
Python 2.7 (32 bit).

(
Python 3.3 currently is not
supported).
Python 2.7
can be downloaded from

http://www.python.org/download/releases/2.7/
.

Notes: Download the 32 bit version

(i.e.,
Windows x86 MSI Installer (2.7) (sig)
)

of Python e
ven if

a 64 bit computer

is used.

T
he
current MS1Probe program relies upon the packages Num
P
y and SciPy which are only
supported as necessary

by

the
32 bit

Python version (
64 bit installations of Python are not
currently compatible with SciPy or NumPy
)

2.)

Download

the SkylineConfig folder from
http://www.gibsonproteomics.org/resources/MS1Probe

.


To
start the
install
ation process
, simply copy
the

downloaded
SkylineConfig folder
preferably
to th
e
C:
\
Program Files
\

directory

(or to the location of your choice)
.

SkylineConfig contains a required report template, compressed copies of NumPy and
SciPy, a copy of SkylineRunner.exe, the script for the tool to be added to Skyline’s Tools
menu, and the c
onfiguration .bat script which will add the script to the tools menu.

3.)


Install

the Python
extension
packages

NumPy and SciPy.

They may be downloaded
from

the following links

http://sourceforge.net/projects/numpy/files/

and

http://sourceforge.net/projects/numpy/files/latest/download?source=files

.
Archived
copies
can also be found in the above downloaded SkylineConfig folder

within

the
Supplemental_NumPy_SciPy

subfolder
.

Aft
er download of the NumPy and SciPy
installer run the corresponding executable file from your computer.

4.)

Close any currently open Skyline documents

(possibly check that you have the most
current Skyline version)
.
C
lick on
the
MS1Probe
Config_
External
.bat
file
located in your
newly generated
folder in
C:
\
Program Files
\
SkylineConfig

or other location you chose
.

Fig.1:




Th
e batch file will

initiate

SkylineRunner

(which is provided to you in the SkylineConfig
folder



which should allow you to perform the below steps
, but which can also be
downloaded from Skyline
)
. The batch file will

automatically add a new tool
to
Skyline’s
Tools menu

called
‘MS1Probe
_External

,

and
at the same time
import

a report template
with
the custom report format required for MS1Probe,
“MS1Filtering_Statistics_
10
_2012.
skyr

.
After opening
of Skyline you will now see this
newly inserted MS1Probe tool.

Fig.
2
:


To confirm
the
proper path setups: in

the

Skyline

Menu


C
lick on
Tools



then
select ‘
External Tools
’ in the pull down, then s
elect the newly
inserted

Tool


MS1Probe_External

,

In the new page
that
open
ed
, go to the “Menu contents’ field and click again on
‘MS1Probe_External’

Y
ou should
now

see the following:

Fig.
3
:




Confirm

that the path shown in the command line corresponds to the exact path that you
had initially copied the downloaded SkylineConfig folder to

C
:
\
programs folder
\

SkylineConfig (and thus you will have the correct path to the
MS1Probe.py executable program fi
le
)
.
If the path is not correct,

you can either adjust
it
in the command line or change the folder path for the *.py file.

5.)

For future

editing
of the external
tool,
you can see under ‘Input Report’ that you can
select different Skyline report templates upo
n expanding on the expand arrow (F
ig
ure

4
).

However if you select a different report template

the

current

tool

MS1Probe may not
support the

different input report
(however this could be adjusted within the python
program itself)
.
The current default
Skyline
report format for MS1Probe is
“MS1Filtering_Statistics_
10
_2012”

which was imported into your Skyline file initially
using the batch file (in conjunction with SkylineRunner).

Fig. 4:




Using the tool with a provided Skyline MS1 Filtering test file
:

After these initial set
-
up steps, the MS1Probe tool may now be used.

To generate a
n

MS1Probe
statistical report from Skyline,
unzip and
open
the provided
Skyline

test

file

called

MS1_Probe_test_WT_KO_v3


from the provided
\
SkylineConfig
\
Skyline_test_file folder.


C
lick on the
Skyline
Tools menu
,

and then

select

the tool

MS1
Probe
_External


as shown
above
in
F
ig
ure

2.

Next

Skyline
automatically
generate
s

the custom

*
.csv report formatted according
to the curren
t MS1Probe

input report template
. Wait a few minutes

while Skyline generates the
custom report that is needed for the
MS1Probe python script

(you can follow
the
progress
by
observing the green progress bar in the lower right
of the

Skyline
window
)
.


The automatical
ly started MS1Probe

python script
will prompt you with a new
command
window asking for some sample/replicate information (condition identifier information):

Enter a minimally distinguishing description per ‘condition’, for example for the provided
Skyline file familiarize yourself with the replicate nomenclature
,
there are

three technical
replicates of an
E. coli

wild

type (WT) strain
and
three technical replicates of an
E. coli

knockout
(KO) strain
.
To review initially what to enter:
go back to

Sk
yline to Menu


Edit


Manage
Results to see replicate naming


In this case distinguishing
identifiers

(case sensitive)

would suffice as WT and KO

to group the
replicates that belong together
, and prompted questions

in the new window

can be answered
for
this example
as shown below:


An additional question as shown above will ask to
append

a description to the base name (name
of the S
kyline file
) when generating the outputs for the statistical report and the original Skyline
custom report (for this ex
ample append ‘test’). The

MS1Filtering Statistics report generated by
the Python Script

will then be opened automatically
. Both
generated *.csv
files will be written to
the same
source
directory
of the processed

Skyline file.

The statistical report provides calculations for each of the 3 precursor isotopes, M, M+1, M+2
(rank 1, 2, 3, respectively) for WT peak area mean (including standard deviation and CV) and
KO peak area mean (including standard deviation and CV). In additio
n, ratios as defined above
by the user are calculated, i.e. ratio of peak area means
for
KO/WT with statistical significance
measures such as a Student’s t test
p

value and a further adjusted, false discovery based
p

value,
referred to as
q

value (
Storey,
J. D. and R. Tibshirani (2003). "Statistical significance for
genomewide s
tudies." Proc Natl Acad Sci US
A 100(16): 9440
-
9445
). An example is shown
below for 1 peptide.


In excel filtering for the top ranked precursor (rank 1, column C), in this case pr
ecursor M, will
simplify the obtained results… However, availability of data for the first 3 precursor isotopes
would allow for addressing possible interferences by selecting the secondary ranked precursor
ion.


For troubleshooting contact Birgit Schillin
g or Alexandria D’Souza

at the Buck Institute for Research on Aging.



Acknowledgment.

All publications that utilize this software

MS1Probe

should provide acknowledgement to
the
Buck Institute for Research on Aging
,

the Gibson Laboratory/Chemistry

Core and refer to the
following website link (
http://www.gibsonproteomics.org/resources/MS1Probe
).