The MALDI mass spectra can :



Identify the molecular mass of the monomer unit of a species ionised
Give an indication for the endgroups of this species
Calculate the polymer distribution parameters for this species
You need :
-
A solvent solubilizing your sample (dichloromethane, THF, DMF, …)
To know the average molecular weight of your sample to choose the best calibrant
Very clean solvent (SPS)
Diluent for standards : acetonitrile/ultrapure water/trifluoroacetic acid (30:70:0.1)
Diluent for matrix : acetonitrile/ultrapure water/trifluoroacetic acid (50:50:0.1)
Eppendorf(s) 1 ml
Micropipettes
A very clean 386 well sample plate
Additionnal notes :
1. Working solutions should be prepared fresh prior to experiments and should be discarded after
use. Do not store for later use, as solutions will degrade in time.
2. Matrix solutions performance may degrade in time. If stored at room temperature and protected
from light, they can be used for up to one week.
3. The lifetime on the sample plate is usually less than 24 hours.
Preparation for polymer analysis : Examples
Example 1
Sample: 10 kg/mol of PMMA, soluble in THF with dithranol (M = 226g/mol)matrix and LiCl.
Prepare three solutions:
- 5 mg/ml of polymer in THF -> 500 µM
- 10 mg/ml of matrix in THF -> 44 mM
- 0.1 mol/L LiCl in THF -> 100 mM
Spotting protocol:
1. Premix sample, matrix and salt solutions volumes 10:10:1
Resulting in a solution : polymer 238 µM, matrix 21 mM, salt 4.77 mM
2. Spot approximately 1-4 µl premixed solution on to the sample plate and allow to dry
With 4 µL solution spotted : polymer 952 pmol, matrix 84 nmol, salt 20 nmol ( 1 : 89 : 20 )
Example 2
Sample: 3 kg/mol of PLA, soluble in THF with DCTB matrix and LiI.
Prepare three solutions:
- 1,8 mg/ml of polymer in THF -> 600 µM
- 15 mg/ml of matrix in THF -> 60 mM
- 0.9 mg/ml LiI in THF -> 7 mM
Spotting protocol:
1. Premix sample, matrix and salt solutions volumes 1:9:1
Resulting in a solution : polymer 55 µM, matrix 55 mM, salt 636 µM
2. Spot approximately 1-4 µl premixed solution on to the sample plate and allow to dry
With 4 µL solution spotted : polymer 220 pmol, matrix 220 nmol, salt 2.5 pmol ( 1 : 1000 : 0.01)
For other examples : see the notebook
Calibrant: choose the appropriate calibrant corresponding to the molecular masses you analyse. Use the
same matrix as the one used for your samples. See the Maldi TOF Mix document or the paragraph below
for explanation. You have to spot the calibrant with the same method as described before.
Additionnal notes :
Spotting protocol B: each solution can be spotted using the dried droplet method (1 µL of polymer
solution + 1 µL of matrix solution on target + 1 µL of salt solution on target). Be careful to keep a ratio
matrix : polymer from 10 to 1000.
Spotting protocol C: for a sensitivity testing, standards (BSA working solutions) can be spotted using a
pre-coated method. Pipette 1 µL of matrix solution onto the sample well. Leave for a few seconds and
then carefully aspirate the matrix solution back into the pipette tip from the edge of the droplet. Discard
this solution to waste. Allow the pre-coated spot to dry and form a thin film of matrix. Pipette 1 µL of
sample solution on top of the pre-coat spot. Immediately spot 1 µL of matrix onto the sample spot. Allow
the sample to dry.
Preparation of the calibration solutions :
The Maldi TOF Mix kit from LaserBio Labs is a specifically solution kit for testing Shimadzu Biotech Axima
series.
Maldi TOF Mix contains calibration vials:
-TOFMIX eight peptides calibration standards
-P14R standard
-Glu1-Fibrinopeptide B standard
-Cytochrome C standard
-Bovine Serum Albumin standard
Calibration standards solutions (stocking solution)
Add 50 µL of standard diluent to each calibrant tube, to reconstitute a stock solution at 5 pmoles/µL
Calibration standards solutions (working solution)
Dilute 2 µL of stock solution in 23 µL of standards diluent (dilution 1 : 12.5). All peptides will be at a final
concentration of minimum 4 pmoles/µL.
- P14R standard
The working solution is the stocking solution : 5 pmoles/µL
- Glu1-Fibrinopeptide B standard
Dilute 10 µL of stock solution in 90 µL of standart diluent (1:10 dilution). Concentration of 500 fmoles/µL
- Cytochrome C standard
The working solution is the stocking solution : 5 pmoles/µL
- Bovine Serum Albumin standard
Dilute 5 µL of stock solution in 45 µL (1:10 dilution)standards diluent. Concentration 500 fmoles/µL
Matrix solution preparation (working solution)
In 1 ml eppendorf tube for each matrix, charge respectively with :




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5 mg CHCA matrix (M = 189.17 g/mol) + 1 ml diluent resulting in a 5 mg/mL solution = 26 mM
20 mg SA matrix (M = 224 g/mol) + 1 ml diluent resulting in 20 mg/mL solution = 89 mM
10 mg DHB matrix (M = 154 g/mol) + 1 ml diluent resulting in 10 mg/mL solution = 64.9 mM
16 mg DCTB matrix (M = 250 g/mol) + 1 ml diluent resulting in 16 mg/mL solution = 32 mM
15 mg HABA matrix (M = 242 g/mol) + 1 ml diluent resulting in 15 mg/mL solution = 89 mM
Spotting protocole : in each well spot 1-4 µL of matrix then 1-4 µL of standart calibration working
solution.
Polymer experiment :
This section provides methods on how to obtain a polymer spectrum using the Axima Confidence.
Run the software
Switch on the computer, Launchpad window will appear. Click to Maldi-MS icon. Some windows appear :
camera view, instrument status and Maldi-MS (at least).
Figure 1 : Launchpad
Insert the sample plate
Go to Acquisition > Exp. Tech. and click to Open door. Choose your tuning mode (reflectron for polymers
and oligomers or linear for polymers) and your mass range : 1.0-10000. Max Laser rate : 50. Be carefull,
the corner cuted of the plate is not the same as the plate described in the picture below, but A1 is well
on the top right edge (Figure 2).
Figure 2 : How to open the door
Figure 3 : Indexing a standart 386 well sample plate
Instrument status shows the vacuum state (Figure 3). It will take around 20 minutes to stabilize. Press to
Operate button before acquisition of a sample in Acquisition > Exp. Tech.
Figure 4 : Instrument status when sample plate just inserted
Figure 5 : Instrument status when ready for experiment
In the region of the plate xy stage appears. If not, the spectrometer has problems to recognize the plate
(or you modified it). We normally use a plate with 96 wells. Check the reference of plate with the
number : DE1487TA. To indicate the plate change, go to Instrument > Acquisition > Slide > Load and
choose your plate (as indicated in the figure below).
Figure 6 : Change plate carrier
If the problem persist, you have to align manually the plate. Press the Align plate button. Moove on the
plate to indicate when you are in A1, A12 and H12 and doubleclik to update informations.
Figure 7 : Align plate
Collecting data from a sample
If you’re not satisfy with your data collection, please check the chapter Peak Processing and be sure that
your spectrum can’t be optimized. Use the settings in the image below for power, profiles and shots.
Select the well you want to analyse by clicking on the plate. Vary the laser power to get a non-saturated
spectrum with good signal-to-noise ratio in the expected mass range. Press Fire and move the laser with
the camera.
Figure 8 : Sample acquisition
Figure 9 : click right to Goto well
Figure 10 : Moove the position of the laser firing by moving in the camera
Peak Processing
You will need to use trial-and-error to establish wich parameters work best with your data. Use the
parameters below as a starting point.
 Reflectron mode (polymers and oligomers):
Scenario: “advanced”
Peak width: 3 to 7 channels
Smoothing method: Gaussian
Smoothing filter width: 3 to 7 channels
Substract base line: unticked to start with
Baseline filter width: greater than 500 channels
Peak detection method : Threshold -25% Centroid
Threshold type: use adaptive threshold (second ration button)
Threshold offset: 5mV
Threshold response: 15
 Linear mode (polymers):
Scenario: “advanced”
Peak width: 10 to 50 channels
Smoothing method: Average
Smoothing filter width: 10 to 10 channels
Substract base line: unticked to start with
Baseline filter width: greater than 1000 channels
Peak detection method : Threshold - Apex
Threshold type: use adaptive threshold (second ration button)
Threshold offset: 5mV
Threshold response: 15
Figure 11 : Peak Processing parameter for linear mode
You wil need to experiment with these settings to establish the best parameters for your samples. As
examples of processing spectra see figures below.
Figure 12 : Smoothing filter 10 and 10 000
Figure 13 : Substract baseline ticked and unticked
When the acquisition is finished, don’t forget to save you analyse File > Save As.
Calibration
Before analysing a sample for molecular weight information the Axima instrument must be calibrated
with a suitable calibrant compound (or mixed calibrants) covering the mass range of interest. This
calibrant sample is applied to the sample stage as with any other sample and a number of shots at the
sample are to obtain a characteristic spectrum (see paragraph Polymer experiment).
First make the acquisition of your calibration well, collect a number of profiles to obtaon a characteristic
spectrum of the reference compound. To use the calibration window, select Calibration from the
Processing menu.
Figure 14 : Calibration window
A calibration reference file contains a list of reference masses which can be used to calibrate the
instrument. For best results the reference masses should span the mass range in which the instrument is
to be used. Click to List references and select the best calibrant file. For example : TOF 2 mixture (figure
15).
Figure 15 : TOF2_mixture reference
First check that the masses displayed in the list are those required. Any reference masses not expected in
the calibrant sample can be deleted using the Delete button. In the figure below, the corresponding mass
of 1047 doesn’t exist anymore. Click to the mass you want, example : 757. In the window 757.8668
appears after Mass: square.
Figure 16 : Select a mass peak
Use the cursor to select the mass corresping in the spectrum. And click to Cursor mass. The value apears
in the square and in the list. Change the tolerance value (50 in the screen shot).
If only one calibrant reference is present on the calibrant sample spot then an option has been provided
to allow the calibration to b forced to Fit Through zero (zero mass at zero time).
Figure 17 : Cursor mass insertion
Do it again with the other values. If the calibration is acceptable and the date used for the calibration
was stored then if the calibration is successful the calibration will be automatically saved. Save your
calibration with a new name. Click to calibrate.
Display options
Acquisitions are stored in diffrents types of spectrum : Profile, Average, Process and Peaks. Process
spectrum is corresponding to the parameters you apply in the paragraph Peak Processing. By acquiring
data, you should at least observed Profile (acquisition in real time), Average (full data) and Process. Click
to the icon Spectrum contents and choose the traces. Note that labels will appear only on the Process
and Peak traces.
Figure 18 : spectrum contents window
By the same window you can choose to show more than one acquisition. In this configuration, two
configurations are possible : Overlay or Stack.
Figure 19 : Overlay configuration
To modify the auto generated labels, please go to View > Display Options > Peak Labels.
Figure 20 : Display options
To add a label corresponding to a peak on the spectrum, click with the middle mouse button on the left
bottom of the peak and again on the the right top of the peak. A square region appeared (see figure
below). Click right, a menu appears. Go to Peak labeling > Add peak. The cursor configuration in still on.
To remove the cursor, click to the off cursor button.
Figure 21 : Add peak in the spectrum