Mass Informatics Platform for Protein Characterization ...€¦ · BPF –Intact Analysis Homepage...

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BioPharma FinderMass Informatics Platform for Protein CharacterizationNative Intact Analysis and Intact Deconvolution in Chromeleon

Step by Step Example – Native Intact Analysis

BPF – Software Homepage

Click on Protein

Sequence Manager

BPF – Protein Sequence Manager

Click on New to

add the protein

Sequence

Click on Import

Protein Sequence

Type or paste protein

sequence here

Max window if

resolution on computer

is less than

recommend.

BPF – Protein Sequence Manager- Intact mAb

Intact analysis the target protein mass

MUST match the value that is

deconvoluted in order of the software to

annotate the masses.

Add disulfide bonds by left mouse

button click on a cysteine and

then link to another cysteine.

Linking dd bonds is

ONLY required for intact

analysis and is NOT

required for peptide

mapping.

If you do not want to link all of

the dd bonds you can create a

custom modification that

correct for the mass difference,

see next slides

All of dd bonds are

linked and the target

protein mass is correct.

If you do not want to link all of

the dd bonds you can create a

custom modification that

correct for the mass difference,

see next slides

Do not link dd bonds and use

variable modification to

correct mass

Add custom modification for

16 dd bonds. For each dd

bond the target protein

losses 2 Hydrogens.

Select any cysteine in the

protein sequence and add the

new DD 16 modification and

the target mass will adjust.

Search the CHO

N-Glycan

database.

Or you can add these pairs of

common glycans to the software

and search them.

A third option for glycans searching would be

to select the Glycans from this list (click on the

define modification list) and then add these as

variable modifications. This list contains all of

the glycans in the CHO or Human database.

Use these glycans for intact

and top down workflows,

however they can not be

used for peptide mapping

searches yet.

A third option for glycans searching would be

to select the Glycans from this list (click on the

define modification list) and then add these as

variable modifications. This list contains all of

the glycans in the CHO or Human database.

Use these glycans for intact

and top down workflows,

however they can not be

used for peptide mapping

searches yet.

BPF – Homepage

Click on Intact

Protein Analysis

BPF – Intact Analysis Homepage

1. Name

experiment

2. Add

Raw Files

3. Assign

conditions

4. Select

Protein

Sequence

5. Select

process

method6. Edit Method

to review

parameters

4. For multiple file experiments choose between batch

processing (results are saved for each individual raw

file) or multiconsensus (results are saved in a

multiconsensus report).

Tip: There is a file size limit for multiconsensus

experiments, 10 raw files, however batch

processing can handle 1,000’s of files.

Tip: There is a file size limit for multiconsensus

experiments, 10 raw files, however batch

processing can handle 1,000’s of files.

Tip: Conditions are helpful when you would like to compare

the results from different type of studies. For example, if

you have 3 files from a control group and 3 files from a

stressed condition, you could assign the appropriate

condition to each file. When processed using

multiconsensus format the software will group the results by

condition making it easy to see differences.

Tip: In the intact analysis workflow the user may select up to 10 different

protein sequences to be searched for annotation. This is not the case for

peptide mapping, only 1 protein sequence is selected. The reason the user

would want to select more than 1 sequence is because the target protein

mass (all chains in the sequence) is used for searching in the intact. So if I

wanted to search for the light chain and heavy chain of a mab I would need

to create 2 separate protein sequences (1 LC and 1 HC) for intact analysis.

For peptide mapping they can be saved in the same sequence.

BPF 2.0– Intact Analysis Homepage- Processing Method

Tip: The following default

methods are provided.

Tip: Notice methods are divided up

by the deconvolution algorithm

ReSpect (isotopically unresolved)

and Xtract (isotopically resolved).

Tip: Source Spectra Method is new to this version of the software and designed to assist the user with the

different options for creating a source spectra. The source spectra is what is deconvoluted to generate the results.

The software provides 3 different ways of generating a source spectra

1) Average Over Selected Retention Time

1) Generate the source spectra by selecting a single scan or average across an user defined time range.

2) Sliding Windows

1) Generate the source spectra by using the sliding window algorithm. User definable window widths

generate average spectrum over a defined time range, each average spectrum is deconvoluted, then

all of the individual deconvoluted spectra are merged together into 1 finial result spectrum

3) Auto Peak Detection

1) Generate the source spectra by using large molecule chromatographic peak detection algorithm.

Tip: Auto peak detection method can only

be processed by adding the experiment to

the queue. You are NOT able to do Manual

Processing for these types of experiments.

BPF – Intact Analysis Homepage- Processing Method

Tip: Enable Automatic Sliding Window Parameter Values

When the click box is checked the software will read the raw data files and provide some recommend

sliding window parameters. These parameters include the Target Avg. Spectrum Width, Target Avg.

Spectrum Offset either scan offset or % offset.

It is recommended that you still adjust the RT Range so that you are setting this time to when you

components of interest are eluting, this will ensure the best optimized parameters.

Uncheck this feature if you want to software to not recommend values for these parameters.

Edit Method or select

Manual Process

BPF – Edit Processing Method (Parameters)

Review parameters

Chromatogram Parameters

CHECK the mz range parameter.

This is also used for the

deconvolution.To see the source spectrum

click on the chromatogram.

This will help you to determine

the best value for the mz

range.

Tip: Salt

Updated m/z range

for better range for

this data set.Tip: Salt peak

disappears

because of the

m/z range

change.

Here you can see the different

source spectra methods and their

parameters. In this example

sliding window is being used.

BPF – Edit Processing Method- Sliding Window

Target Avg

Spectrum Width

Target Avg

Spectrum Offset

Window 1 deconvoluted

spectrum

Merged results spectrum

Average source

spectrum is generated

for the spectrum width,

then deconvoluted

The individual deconvoluted spectra are merged together to create the finial

deconvoluted result. The merge parameter are used to achieve the finial result.

BPF – Edit Processing Method- Sliding Window

How the software determines the recommended Spectrum Width?

The sliding window width is determined by calculating the

autocorrelation function of the chromatogram and examining this to

determine the characteristic scale width of peaks in the

chromatogram. This approach proved significantly more robust, more

objective, less sensitive to parameter choices, less sensitive to the

baseline, and less sensitive to the peculiarities of individual features in

the chromatogram than attempting to identify and examine some subset

of the chromatographic peaks themselves.

What is the difference between the scan offset and the % offset? 1) In Scan Offset mode, each window is offset from its predecessor by

the user-specified number of scans – i.e. if the first window began at

scan 127 and the scan offset was 2, the second window would begin at

scan 127 + 2 = 129. In Percent Offset mode, each window is offset from

its predecessor by a the user-specified percentage of the window width

– i.e. if the first window began at an RT of 2.5 min, the window width

was 0.2 min, and the percent offset was 25%, the second window would

begin at an RT of 2.5 + 0.2 * 0.25 = 2.55 min.

Sliding window automatic parameters

BPF – Edit Processing Method- Auto Peak Detection

Toggle to auto peak detection and

you will notice the blue area shading

appears on the chromatogram trace.

BPF – Edit Processing Method- Average Over RT

Toggle to Average Over Selected

Retention Time. You can enter a

retention time range here and the

plot will create an average spectrum

on the left.

Red box is the RT range

used in the parameters. You

can also click on the

chromatogram directly like

done in Qual Broswer.

You can toggle between

averaging and zooming

of the chromatogram

using these buttons.

BPF – Edit Processing Method

Can you also view the

other raw files.

BPF – Edit Processing Method- Deconvolution

You can select the different

Deconvolution Algorithms. This design

allows the user to toggle between the

different algorithms easily.

BPF – Edit Processing Method- Deconvolution- Xtract

MZ range is used for

deconvolution from the

chromatogram parameters.

Xtract is used for Isotopically

resolved data sets only.

Tip: Pay attention to the

charge range especially if you

deconvoluting MS2 spectra

for top/middle down. You will

want to set this charge range

to 1 to 50 (or maybe less).

Tip: For MS2 spectra

deconvolution set this value to

1 to generated results.

Tip: If you are seeing the

value off by 1 Da, try turning

off the consider overlaps

BPF – Edit Processing Method- Deconvolution- ReSpect

MZ range is used for

deconvolution from the

chromatogram parameters.

ReSpect is used for Isotopically

unresolved data sets only.

This is the output mass range for the results of the deconvolution.

This is different than the model mass range as it does not affect the

peak model used in the deconvolution like changes the model mass

range. Therefore use this feature to narrow the deconvolution

spectrum for display purposes without affect the results.

Resolution is also a very important parameter for ReSpect and is used in

determining the mass peak model.

Here you have 2 options…

1. Raw file specific

1. The software will read the value from each raw file and use that

value.

2. This information is not displayed here…. But the individual

values can be found under the parameters-> Save Method

Report (see below)

2. Method specific

1. The software will read the resolution from the first file in the data

set and apply this value to all of the raw files.

See next slide for explanation.

Mass Probability Distribution Profile (legacy plotting)

Isotopic Profile (new plotting)

Mass Probability Distribution (legacy)

The Mass Probability Distribution shows the

probability that a component has a particular

average mass multiplied by its abundance. If all

the masses could be determined precisely, this

would be a centroid spectrum. In practice, it

becomes a set of Gaussian peaks, with a widths

proportional to their uncertainties – typically this

will be on the order of a few ppm.

Isotopic profile

The IsotopicProfile shows the isotopic distributions

for all the components identified by the

deconvolution plotted vs mass. This is what peaks

in the original m/z spectrum would ‘look like’ if

they were multiplied by their associated charge

states and plotted vs mass. This Isotopic Profile

spectrum can be used for visual comparison with

the original m/z spectrum to examine how peaks

were assigned by the deconvolution. Note that the

Isotopic Profile spectrum does not include the

background signal. The background signal is

identified and removed as part of the

deconvolution process.

Tip: If you process your data file using

the default parameters and you do not

get any results, start with optimizing this

parameters. I set these values to 4 to 4,

if no results are present.

Tip: For NATIVE data you MUST check the

minimum adjacent charges and set this value to

4 to 4 because native data does not have an

many charge states as reduced samples.

Model mass range should be set around your target mass. For the subunit

example the target mass is ~25,000. Therefore I will set this value to 20,000

to 28,000. Or if I am looking at an intact mAb molecule where the target

mass is 148,000, then we can set this range to 140,000 to 150,000. It is

important to understand this parameter does not just defined the x-axis of

the deconvoluted spectrum, but it is used for the peak mass model.

Minimum adjacent charges parameter works directly with model mass

range. The number enter here is 6 to 10 as a default. What this means is

the lower model mass (10,000) will require a minimum of 6 adjacent charges

and the upper mass (160,000) will require a minimum of 10 adjacent

charges. And all masses in-between will be on a linear line for required

adjacent charges.

Set the target mass as close to the value of what you think your mass will

be. It does not have to be exact but it should be close. If you have an

mixture in the sample (Light and heavy chain) you can set this value to

something in the middle.

BPF – Edit Processing - Chromatogram/source spectrum

You can click on the chromatogram just

like in Qual Broswer to create an

average source spectrum and/or zoom

in or out.

Use the mode buttons to

change between averaging

and zooming.

View the chromatogram and

source spectrum for multiple

files.

Tip: This window can be floated to

allow you to see the images better.

Right click for some additional

options.

BPF – Edit Processing Method- Identification

If you have selected protein sequences on the intact homepage

and then edited the method you will see protein here. The

protein sequences doe not get saved with the processing

method so you will need to make sure you select the proteins

you are interested in using on the intact home page.

BPF – Edit Processing Method- Identification

This parameter is a mass tolerance for sequence matching. For

example, if the mass of the light chain sequence is 23,428.524 and I

set this value to 20.00 ppm that means the software will try to match

the deconvoluted results using 20 ppm window of the theoretical

value.

Tip: Sometime you might need to increase this value to

30ppm if you are not seeing all of the different glycoforms form

intact mAb. What I find helps, it to search with the default

value, then if I am missing something I will increase to value to

30ppm and see how off the match using the Matched Mass

Error in the main results table. If I see it is only 23 ppm them I

might try to optimize some of the reconvolution parameters, or

my source spectrum to improve the results.

BPF – Edit Processing Method- Identification- DAR

To enable the average Drug to Antibody

Ratio (DAR) check this check box.

BPF – Edit Processing Method- Identification- DAR

Next you will need to select modification from the drop down list. This

is require for the software to determine the average DAR.

BPF – Edit Processing Method- Identification-DAR

First the software will look for annotations

that contain the selected modification.

Tip: The average DAR value is determined using the

equation above. This value will be on the deconvoluted

spectrum and in the DAR tab. The user can override the

software and change the drug load value if needed and the

user can also select which identifications are used in the

calculation. This guide is for subunit analysis and will not go

into these details so see the other DAR guide for more

information.

Average DAR:= (sum (drug load*intensity) / sum intensity)

The drug load is automatically determined by looking at the number

of the modifications that are assigned to the identification.

BPF – Edit Processing Method- Identification- Multiconsensus

Tip: Minimum number of required occurrences means how

many files do you want to require the component to be

present. For example, if I had 10 raw files and I only wanted

to see all of the component present in all of the files and

nothing else, then I would set this value to 10. But if I want

see component that are present in some samples but not the

other then this value should be set as a limit. Remember

this is a minimum.

These parameters are used to merge together the deconvoluted

results from multiple files into 1 results. When the user selects

multiconsensus for the result format on the intact homepage, the

software will merge together the results into 1 result. I like to think

about this as if I was doing this in Excel (like you had to in the older

version of the software). If you had 3 raw files, you would process

them all individually and then export the results to excel. Next you

would put them together in one list. You would group them together

by mass and retention time. If the values were within some limit that

you define then you would say those masses are the mass species.

The software is doing the same thing just automatically. It will

process each file using the parameters you define and then merge

together the results using these parameters.

BPF – Edit Processing Method (Parameters)-Report

This tab contents the basic

reporting features that were in the

older software. Tip: This basic report will only work with

single file experiments. It does NOT work

for multiconsensus or DAR experiments.

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: Right click to export

summary into Excel or

You can overwrite methods

(except for the default methods)

and experimental results.

BPF – Intact Analysis Homepage- Average over RT

Add to queue for

automatic processing Or manual processing

Both batch and multiconsens result format are

available for this processing method.

Assign conditions for easier

comparison or to see %CV in the

results table to help assess

reproducibility.

BPF –Manual Processing- Average over RT

Main page when you

open your results or do

manual processing

Tip: Real time optimization has

all of the parameters you need to

fine tune the values.

Float the windows so you can

see the images better for

these large experiments.

Adjust the ReSpect deconvolution

parameters.

New parameters

Default parameters

All 10

chromatogramsSource

spectrum

Annotated deconvolution

spectrum from all 10 raw

Inactive table

Raw file specific

information

%CV shows the

reproducibility.

Annotated deconvolution

spectrum from all 10 raw

This view allows for a quick

comparison across all 10

files.

Right click to

export results

At the raw file level you can

add the deconvoluted

spectrum to the library.

You can select any 2

spectrum from the table

above to compare.

Right click for

other options.

BPF –Manual Processing- Sliding Window

Which over to

sliding window Adjust RT

Sliding window

running live.

Sliding window

running live.

Deconvoluted

spectrum for the

current window

Source spectrum

for the current

window

Sliding window

results.

Raw file level

information show the

abundance trace.

Change the sequence from

variable glycans list to CHO

Press edit

Add the CHO sequence and

remove the variable glycans to

the experiment.

Go back to process and review

to reprocess using the smaller

protein sequence.

Results search CHO

database

Creating Workbook and using Chromeleon

BPF – Creating intact workbook

Select components of interest to

be monitored in Chromeleon and

right click, Save As Intact

Workbook

BPF – Creating intact workbook

Save workbook in BPF.

BPF – Intact workbook Manager

Saved workbook are stored and

managed from within BPF in this

new tab. Select the workbook of

interest and export to a fomat

that can be imported into

Chromeleon 7.2.9

BPF – Intact workbook Editor

Review the parameters and

delete the components in the

workbook from this editor page.

You can also export to

Chromeleon format from her as

CM 7.2.9 – Intact Protein Deconvolution

New Intact Protein

Deconvolution processing

inside of Chromeleon 7.2.9

CM 7.2.9 – Process Method Selection

CM 7.2.9 – Processing

CM 7.2.9 – Deconvolution Results

CM 7.2.9 – Custom Reporting

BPF 3.1 & CM 7.2.9 –Targeted Component

CM 7.2.9 – Define Component Detection and Define Scoring

CM 7.2.9 – Score All Injections for Targeted Components

Mass Informatics Platform for Protein Characterization ...€¦ · BPF –Intact Analysis Homepage...

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