The world leader in serving science
BioPharma FinderMass Informatics Platform for Protein CharacterizationNative Intact Analysis and Intact Deconvolution in Chromeleon
The world leader in serving science
Step by Step Example – Native Intact Analysis
3
BPF – Software Homepage
Click on Protein
Sequence Manager
4
BPF – Protein Sequence Manager
Click on New to
add the protein
Sequence
5
BPF – Protein Sequence Manager
Click on Import
Protein Sequence
Tip:
Type or paste protein
sequence here
Tip:
Max window if
resolution on computer
is less than
recommend.
6
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.
Tip:
Linking dd bonds is
ONLY required for intact
analysis and is NOT
required for peptide
mapping.
7
BPF – Protein Sequence Manager- Intact mAb
Tip:
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.
8
BPF – Protein Sequence Manager- Intact mAb
Tip:
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.
9
BPF – Protein Sequence Manager- Intact mAb
Select any cysteine in the
protein sequence and add the
new DD 16 modification and
the target mass will adjust.
10
BPF – Protein Sequence Manager- Intact mAb
Search the CHO
N-Glycan
database.
Or you can add these pairs of
common glycans to the software
and search them.
11
BPF – Protein Sequence Manager- Intact mAb
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.
Tip:
Use these glycans for intact
and top down workflows,
however they can not be
used for peptide mapping
searches yet.
12
BPF – Protein Sequence Manager- Intact mAb
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.
Tip:
Use these glycans for intact
and top down workflows,
however they can not be
used for peptide mapping
searches yet.
Save
13
BPF – Protein Sequence Manager
14
BPF – Homepage
Click on Intact
Protein Analysis
15
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.
16
BPF – Intact Analysis Homepage
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.
17
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.
18
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.
19
BPF – Intact Analysis Homepage
Edit Method or select
Manual Process
20
BPF – Edit Processing Method (Parameters)
Review parameters
Chromatogram Parameters
21
BPF – Edit Processing Method (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
peak
22
BPF – Edit Processing Method (Parameters)
Updated m/z range
for better range for
this data set.Tip: Salt peak
disappears
because of the
m/z range
change.
23
BPF – Edit Processing Method (Parameters)
Here you can see the different
source spectra methods and their
parameters. In this example
sliding window is being used.
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.
24
BPF – Edit Processing Method- Sliding Window
Target Avg
Spectrum Width
Target Avg
Spectrum Offset
1
Window 1 deconvoluted
spectrum
2
3
4
5
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.
25
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
26
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.
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.
27
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.
28
BPF – Edit Processing Method
Can you also view the
other raw files.
29
BPF – Edit Processing Method- Deconvolution
You can select the different
Deconvolution Algorithms. This design
allows the user to toggle between the
different algorithms easily.
30
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
31
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.
32
BPF – Edit Processing Method- Deconvolution- ReSpect
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.
33
BPF – Edit Processing Method- Deconvolution- ReSpect
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.
34
BPF – Edit Processing Method- Deconvolution- ReSpect
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.
35
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.
36
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.
37
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.
38
BPF – Edit Processing Method- Identification- DAR
To enable the average Drug to Antibody
Ratio (DAR) check this check box.
39
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.
40
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.
41
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.
42
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.
43
BPF – Edit Processing Method (Parameters)
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.
44
BPF – Edit Processing Method (Parameters)
Tip: Right click to export
summary into Excel or
Word.
You can overwrite methods
(except for the default methods)
and experimental results.
45
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.
46
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.
47
BPF –Manual Processing- Average over RT
Float the windows so you can
see the images better for
these large experiments.
48
BPF –Manual Processing- Average over RT
Adjust the ReSpect deconvolution
parameters.
49
BPF –Manual Processing- Average over RT
New parameters
Default parameters
50
BPF –Manual Processing- Average over RT
All 10
chromatogramsSource
spectrum
Annotated deconvolution
spectrum from all 10 raw
files
Inactive table
51
BPF –Manual Processing- Average over RT
Raw file specific
information
%CV shows the
reproducibility.
Annotated deconvolution
spectrum from all 10 raw
files
This view allows for a quick
comparison across all 10
files.
52
BPF –Manual Processing- Average over RT
Right click to
export results
At the raw file level you can
add the deconvoluted
spectrum to the library.
53
BPF –Manual Processing- Average over RT
You can select any 2
spectrum from the table
above to compare.
Right click for
other options.
54
BPF –Manual Processing- Sliding Window
Which over to
sliding window Adjust RT
Range
55
BPF –Manual Processing- Sliding Window
Sliding window
running live.
56
BPF –Manual Processing- Sliding Window
Sliding window
running live.
Deconvoluted
spectrum for the
current window
Source spectrum
for the current
window
57
BPF –Manual Processing- Sliding Window
Sliding window
results.
58
BPF –Manual Processing- Sliding Window
Raw file level
information show the
abundance trace.
59
BPF –Manual Processing- Sliding Window
Change the sequence from
variable glycans list to CHO
Press edit
60
BPF –Manual Processing- Sliding Window
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.
61
BPF –Manual Processing- Sliding Window
Results search CHO
database
The world leader in serving science
Creating Workbook and using Chromeleon
63
BPF – Creating intact workbook
Select components of interest to
be monitored in Chromeleon and
right click, Save As Intact
Workbook
64
BPF – Creating intact workbook
Save workbook in BPF.
65
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
66
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
well.
67
CM 7.2.9 – Intact Protein Deconvolution
New Intact Protein
Deconvolution processing
inside of Chromeleon 7.2.9
68
CM 7.2.9 – Process Method Selection
69
CM 7.2.9 – Processing
70
CM 7.2.9 – Deconvolution Results
71
CM 7.2.9 – Custom Reporting
72
BPF 3.1 & CM 7.2.9 –Targeted Component
73
CM 7.2.9 – Define Component Detection and Define Scoring
74
CM 7.2.9 – Score All Injections for Targeted Components