BioPharma Finder 1.0 How to Guide Basic Peptide Mapping · How To Guide Basic Peptide Mapping. ... analysis workflow choose protein ... BPF 1.0 –Peptide Mapping Analysis Homepage

How To Guide

Basic Peptide Mapping

2 Proprietary & Confidential

BioPharma Finder 1.0 – Software Homepage

Peptide Mapping & Intact Analysis both in one software


BPF 1.0 – Protein Sequence Manager

Protein Sequence Manager

• Starting point for the software.

• Database for storing protein

sequences similar to Proteome

Discoverer.

• Both peptide mapping and intact

analysis workflow choose protein

sequence from this central

location.

• Import FASTA file or paste the

sequence into a text box.

• Define multiple chains (e.g. two

light chains, two heavy chains).

• Define disulfide bonds (information

used for intact analysis)

• Choose fixed modifications

• Select variable modifications and

list of possible glycosylation

structures.

• User definable default modification

list.

Sequence database enables user

to only have to load the sequence

once for intact or peptide mapping

experiments.

User definable default

modification list.


BPF 1.0 – Intact Protein Analysis

Intact Protein

Analysis

• Same features currently

available in Protein

Deconvolution 4.0.

• New 64 bit ReSpect .dll

which could provide

improved results for

experiments that contain

dimers but this is still

being tested.

• No other feature

improvements were made

to this workflow.


BPF 1.0 – Peptide Mapping Analysis Homepage

Peptide Mapping

Analysis

Steps to creating a new

experiment.

1. Name experiment

2. Select raw files

3. Choose Protein Sequence

4. Select Processing Method

5. Click “start processing” to

beginning analysis or “edit

method” to review

parameters and then

automatically beginning

data processing.

Condition assignment

for multiple raw file

experiment which

makes data

interpretation easier.

Batch processing using

template methods to

increase throughput.

Default processing

method to assist users

with different workflows.

1

2

3

4

Edit Processing Method to

review parameters and

double check Threshold

setting along with other

parameters.

5


BPF 1.0 – PMA->Processing Method Editor

Peptide Mapping Analysis

Wizard like editor to help with custom

processing methods.

1. Component Detection

2. Identification

3. Save Method and Review Summary

Improved graphics for viewing the

absolute MS Signal Threshold.

Zoom in the y-axis to see where the

red line is located. As you change the

absolute MS Signal Threshold the red

line move up and down.





processing methods.


2. Identification


Improved parameter

design to make it

easier to user to set

conditions.





processing methods.


2. Identification


Export experiment

summary for record

keeping and

reporting.


BPF 1.0 – PMA->Run Queue

The Queue

• Where the user monitors the

experiments process, stops jobs in

progress and opens results.

• User can “queue up” a large

number of experiments at one time

and let them run over night which

increases their productivity.

• Experiments can be actively

processing while the user

simultaneously review results.

• User can “queue up” experiments

and while they are running they

can perform Intact Analysis data

processing.

Completely new feature

for Peptide Mapping.

Allows users to submit

multiple experiments.


BPF 1.0 – PMA->Process and Review

Major improvements to component table,

sortable, filterable, peptide sequence,

modification list, mass error (delta ppm), ID type.

Export and select

component

export to excel

Peptide fragment

map coverage

automatic

generation.

Six different types of plots, multiple raw

file display and all interactive.

Process & Review• Where the user reviews their

results.

• Real Time Optimization

• Adjust parameters real time

and the reprocess

experiment.

• Chromatogram

• Results Table

• Protein Sequence

• Spectra

• Full Scan Spectra

• MS2 Spectra

• Peptide Fragment Coverage

Map

• Floatable windows!



Major improvements to component table,

sortable, filterable, peptide sequence,

modification list, mass error (delta ppm), ID type.

Export and select

component

export to excel

Peptide fragment

map coverage

automatic

generation.

Six different types of plots, multiple raw

file display and all interactive.


results.



and the reprocess

experiment.

• Chromatogram

• Results Table


• Spectra


• MS2 Spectra


Map





results.



and the reprocess

experiment.

• Chromatogram

• Results Table


• Spectra


• MS2 Spectra


Map


Peptide fragment

map coverage

automatic

generation.

Six different types of

plots, multiple raw file

display and all

interactive.

Select different modes of fragment spectra to

display, important for fusion instruments when

use collects data using CID, HCD and ETD.

Experimental MS2

and predicted MS2

spectra


BPF 1.0 – PMA->Mapping Coverage

Coverage• Protein sequence coverage

map

• Chromatographic peak

shading.


Display sequence

coverage

information for

multiple files all in

one place.



Colors are based on protein sequence (key to left). Each

component peak is shading using the appropriate color.

The width and height are specific to each component. The

height of the shading is the height of the component.



The peak at RT 40.43 mins has a component from the light chain sequence and a

small component that is unidentified. Where as the peak at RT 41.83 has several

components from the heavy chain sequence and a small light chain component.

This is NOT absolute quant, but is designed to provide a visual aid to the depth of

our mass spect data. This can also be used to assist user optimizing the

component detection parameters when their chromatography is not perfect!


BPF 1.0 – PMA->Mapping Modification Summary

Modification Summary

• Modification summary report

currently available in

PepFinder.

• New columns in report

including,

• Normalized Time Shift

• Predicted Time Shift

• Peptides

• Sequence

• Confidence

• Comments

• Interactive report- when

user clicks on modification

in the top grid the

components used to

calculate the %

abundance will appear in

the bottom gird.

Removing the black box

for this super important

feature.


BPF 1.0 – PMA->Sequence Variant Example

New column for sequence

variants to enable user to easily

find this specific modifications.

Automatic fragment coverage

map to assist with fast

confirmation of modification.

Display SIC for modification

across multiple files at the

same time.


BPF 1.0 – PMA->Disulfide Bond Example

All peptide involved in the

dd bond are highlighted

on the protein sequence.

Column filters enable the

user to quickly find all

components that were

identified as dd bonds..

Right click options on main table allow user to

• Export all results or selected results to excel.

• Create mgf file for Host Protein searching in

Proteome Discoverer.

• De Novo sequencing


BPF 1.0 – PMA->Large Study Processing

Large Study Example

• External beta tester processed 110 raw files using PepFinder 2.0 and presented the results at ASMS 2015 (excellent poster!).

• Manual inspection extremely difficult and very time consuming.

• Same 110 raw processed using BPF (4 hrs)

• Manual inspection is no longer painful.

• Chromatograms from different groups or all files can be displayed at the same time.

• Conditions are grouped together and average MS area and %CV are calculated.

• Protein coverage maps are easily generated with just a click.

Display BPC for 110 raw

files at the same time.

And you can copy/paste

the image as well.


BPF 1.0 – PMA->What is that Peak?

Not unidentified

because it is not

blue… so what is it?



Most abundant ion

(+2, 676.8972) in not

in the component

table? So why was

this ion missed and

what is it?



Real time optimization allows the user to change

the parameters while looking at the results. The

experiment can reprocessed using the new

values.

With some trial and error I found that

changing the Mass Tolerance from 4

to 7 ppm and adjusting the min peak

width from 0.4 to 0.32 I was able to

detect the component at 767.8792.



Light chain peptide!!! This

peptide was co-eluting with

another peptide and required

very small adjustments to the

component detection

parameters, but without the

chromatographic shading I

would have never knew it

was not identified.

Step by Step Example

BSA Stressed Study


BPF 1.0 – Software Homepage

Click on Protein

Sequence Manager



Click on New to

add the BSA

protein Sequence



Click on Import

Protein Sequence

Tip:

Type or paste protein

sequence here



Double click on

any “C” to add

static

modification

Tip:

When clicking

on a specific

residue click on

the left side of

the letter.

Tip:

Add a category for each

sequence. Example

disulfide bonds, reference,

peptide mapping…

Tip:

Double click on any C to link to another

C to form disulfide bonds. This is

information is not required for peptide

mapping.



Select

Carbamidomethylation

Check

Apply to All



Select Variable

Modifications to

add more mods.

Tip:

Static mods are

now highlighted.



Add Glycation, Oxidation

(MW) and Double

Oxidation from the side

chain modification list.

Tip:

Add default

modification list

in one click



Click Default

Modifications to

view list



Click

Save

Tip:

User can define

their own

default

modifications



Click Save then

Cancel to close

editor and return

to protein

sequence

manager.



Tip:

Protein

sequence is

now added to

the list.

Click Home


BPF 1.0 – Homepage

Click on Peptide

Mapping Analysis


BPF 1.0 – Peptide Mapping Homepage

1. Name

experiment

2. Add

Raw Files

3. Assign

conditions

4. Select

Protein

Sequence

5. Select

process

method6. Edit Method

to review

parameters

Tip:

The component detection algorithm has been improved in this

software compared to PepFinder so you might see

differences. We recommend that you edit the processing

method and use the new BPC chromatogram graphic to view

the absolute threshold value for your data set. If the analysis

is too slow (several hours) stop the analysis and then check

the red line in the parameters to see if it is too low).



Tip:

Uncheck this box to use the values in a saved

process method. When this box is checked the

software will automatically calculate some of the

parameters based on the raw data files (i.e.

threshold, peak width).



Tip:

Click here if you want to load

an experiment that has been

processed already. You can

also open results from the

Queue.


BPF 1.0 – Edit Processing Method (Parameters)

Component detection

tab in the processing

method editor.

Tip: Interactive red line represents the

Absolute MS Signal Threshold value on the

BPC of the first raw file in your list. Zoom in

the vertical direction and then change the

value and watch the red line move.

New parameters for determining MS signal

threshold. The absolute MS Single Threshold is

the product of the MS Noise level (automatically

determined) and the S/N Threshold.



Zoom in the y-axis by

moving the mouse

down in a straight line.



Red line goes down

Adjust the MS Noise level and/or

the S/N Threshold and the

Absolute MS Signal Threshold will

change and the red line will move..

Tip: PepFinder used a set value of 1,000 for the MS

Noise Level therefore if you want to reproduce your

results here is an example. If PepFinder gave a default

MS signal threshold of 20 (meaning 20,000 counts). This

means the program determined the MS noise level is

1000, and then applied a default S/N threshold of 20. In

the new version, the same default parameters will be

written as an MS Noise Level of 1000, and S/N threshold

of 20. The routine for noise level determination was

changed in the new version, that’s why you may see a

different value for the same file.



Tip: If you do not want to see the identifications with the

+ or – a mass value (example 1:V149-

K168=2134.946m(~V149+57.0215) in your results you

can uncheck the “enable mass search for unspecified

modifications” on this page.

Identification parameters are the

same as in PepFinder 2.0 with a few

additions to make it easier to use.

Tip: Non-specific protease can be used for

experiments with multiple proteases or

peptide studies.



Save the processing method so

it can be used later.

Tip: Summary of your processing method

parameters and your experiment if you

selected raw files and a protein sequence.

Tip: If you want to process a new data set

using the save parameters in a processing

method you must uncheck the Enable

Automatic Parameters Values (shown

below) which is on the peptide mapping

homepage where you select your

processing method. When you uncheck

this box, the software will use the

parameters set the method.



Tip: Right click to export

summary into Excel or

Word.


BPF 1.0 – Queue

Run Queue is new for this software and

will help you keep track of your

experiments and is a great place to open

your results.

Tip: Open Results. In this first version of BPF you will not

be able to physically select a location to save your results

like you did in PepFinder. The software automatically

saves the results into the database that is on your computer

and will save the .pmf file in the following location

C:\ProgramData\ThermoScientific\BioPharma\OutputFiles.

This is a feature that we will be improving in next version.

Tip: Once you have submitted an

experiment you can Stop the processing

by clicking here. If you have other

experiments submitted to the queue and

you stop the job that is activating running,

you will need to press RUN to start the

queue back up.


BPF 1.0 – Process and Review

Main page when

you open your

results.

Tip: If you are looking for the

protein coverage map or the

modification summary go to the

Mapping tab.

Enhanced table with filtering, sorting, exporting, peptide sequence, modification, site of modification, ID type, Delta ppm, check boxes

to select specific components to be exported, average Area per conditions and %CV. See next slides for more details.


BPF 1.0 – Process and Review -> Filter

Tip: All of the columns can be filtered

using a large amount of different

functions including custom.

Tip: Click here to see a list of the

different functions for filtering and

multiple filters can be applied at the

same time.

Tip: To sort the identification list starting from the first

residue in the first protein (the same way the results can

be sorted in PepFinder), first filter the Identification column

for (NonBlanks), then sort on the column.


BPF 1.0 – Process and Review -> Sort

Tip: To sort the identification list starting from the first

residue in the first protein (the same way the results can

be sorted in PepFinder), first filter the Identification column

for (NonBlanks), then sort on the column.


BPF 1.0 – Process and Review ->Click

Click on a component and the other

windows will display the results for

that component.Tip: For multiple file experiments the information that is displayed when the user

clicks on a component is from the first raw file in your list. You can see the other

raw file information by 1) clicking on the + in the table and then click on a specific

raw file 2) right click on the Chromatogram and select a different raw file from the

list or select ALL of the files to see the BPC or SIC for a selected component.


BPF 1.0 – Process and Review ->Chromatogram

Right click for chromatogram options.

Select Chromatogram allows the user to

change what plots are displayed, reset

scale when zooming, copy and label using

different options included peptide ID.

Tip: If you zoom in

on any of the

chromatograms you

can double click to

zoom out..



Raw files used in the

experiment will be

displayed here.Six different plots can

be displayed for 1 single

raw file. If you would

like to display

information from more

than 1 raw file you can

only select 1 plot type.



Selected Ion Chromatogram (SIC)

displayed for a single component

from all 4 raw files at the same

time.


BPF 1.0 – Process and Review -> Floatable

Want to see the data better, grab

the window and pull it out! All of the

windows are floatable.


BPF 1.0 – Process and Review -> Chromatogram

SIC for all of the raw files. The

green highlight shows the area

used to determine the Area value in

the table.


BPF 1.0 – Process and Review -> Chromatogram

Base Peak Chromatogram (BPC)

for all of the raw files. The green

highlight shows the area used to

determine the Area value in the

table.


BPF 1.0 – Process and Review->Results Table

Pull the table out for

a close look.



ID Type tells the user how the component was

identified. You will see Full, MS2 or MS2/Full.

The mixture will be shown when you have a

multiple file experiment and some of the raw file

have MS2 but not all of the files.

Delta ppm is the error

between the

theoretical and

experimental

Monoisotopic mass.



Expand to see

raw file specific

information.



Filters



Right click

anywhere to see

other options.

Export all of the results or

export only selected

components..

Create mgf file for host

cell protein searching

using Proteome

Discoverer.De Novo

Searching


BPF 1.0 – Process and Review-> Fragment Coverage Map

Fragment coverage map

is generated

automatically.


BPF 1.0 – Process and Review->Sequence Location

Highlighted peptide

sequence so you can see

where the peptide is in

the protein.


BPF 1.0 – Process and Review->Full Scan

Deconvoluted

spectra

Full Scan

Tip: Double click

to zoom out.


BPF 1.0 – Process and Review-> MS2 Spectra

Experimental

MS2Tip: Double click

to zoom out.

Predicated

MS2

Tip: This is the

parent ion.



If your experiment has different

modes of activation or if you acquired

a single mode of activation at different

resolution all MS2 spectra are

automatically searched and can be

displayed by using these toggles.

Right click for options. You

can adjust the prediction

parameters, hide the

prediction spectra and copy.



Prediction parameters if you want to

adjust the values or see what your

peptide fragmentation would look like

using another mode of activation.



Prediction parameters if you want to

adjust the values or see what your

peptide fragmentation would look like

using another mode of activation.



Prediction HCD

spectra


BPF 1.0 – Mapping-> Sequence Coverage Map

Mapping tab

Protein sequence coverage map

which is for each raw file. We will

continue to improve this feature in

the next versions of the software.

Chromatographic shading and protein

report. If you expand the protein you can

see the protein coverage in the different raw

files. Shading can be helpful in observing

the component detection parameters, and

what you might be missing.



Protein coverage for the

different raw files.



Shading, red are identified

components, green are

unidentified.

Tip: Click on each row to turn on and off

the shading for that row. If you hold

“Ctrl” to show multiple shading colors at

one time.



Right click for

parameters.



Right click for

parameters.



Right click for

parameters.



Zoom in to see the

details of the

shading.


BPF 1.0 – Mapping->Modification Summary

Click here to see

the modification

summary.

This is the same modification summary report that

was generated in PepFinder but now with some great

new features, including filtering, sorting, exporting to

excel, exporting selected modifications to excel and

the component table below shows the components

that are used in the % abundance calculation for each

modification.



Click on a

specific

modification

This table automatically updates to display all of the components that contain the residue that is

modified. The blue components are the ones that are used in the calculation. In future versions of

the software you will be able to select which components you want ot use in the calculation.

Click on a component in the

component table a the plots

will update to display

information for that

component allowing for easy

data review.



Filter using contains

“Glycation” so only the

modifications that contain

glycation are displayed.

Right click to export

checked modifications

to excel.



Experiment

information

Column headers for

the modifications.

They are a little hard

to find and are

grouped together.

Documents

BioPharma Finder 1.0 How to Guide Basic Peptide Mapping · How To Guide Basic Peptide Mapping. ... analysis workflow choose protein ... BPF 1.0 –Peptide Mapping Analysis Homepage