NCBS – MS FACILITY:
PROTOCOL FOR THE DETERMINATION OF INTACT MASS OF
PEPTIDE/PROTEIN SAMPLE
AIM: Determine the intact mass of the Peptide/protein sample.
The process is described in three parts.
First part describes the instrument set up and chemicals required for the analysis.
The second part describes the calibration of the instrument using horse heart Myoglobin.
The third exemplifies the determination of intact mass of a protein sample.
PROCEDURE:
I.
Instrument and Chemicals
Waters Ultima QTOF (former Micromass)
Software - MassLynx 4.1
Figure 1) Waters Q-TOF Ultima mass spectrometer (former Micromass)
Chemicals:
Myoglobin from Equine Heart (Sigma Aldrich Cat#M1182
Water (LC/MS grade-Fluka)
Acetonitrile (LC/MS grade –Fluka)
Methanol (LC/MS grade –Fluka)
Formic Acid (LC/MS grade - Fluka)
Instrument requirements:
1. MS Tune file – C:\qtof_users\karthik.pro\acqudb\protein_hhm_pos100720.ipr
2. Tune parameters are summarized in the following Excel file (QTOF computer -C:\Documents and
Settings\labusers\My Documents\karthik results\MS parameters)
– MS Parameters for reference
3. Put on the API gas(Ultra High pure Nitrogen) and Collision gas( Argon)
4. Change the capillary voltage from 0 to 3.3 and Desolvation temperature from 20 to 200(these
parameters are kept at 0 and 20 when the instrument is idle.
5. 250µl syringe and Infusion pump
II.
Calibration of the Instrument with Horse Heart Myoglobin
A. Prepare the following solutions:
1. Spray mix – 0.1% Formic acid in 50:50 acetonitrile: water
2. Myoglobin stock solution – 100µM in water(stored in -20C)
3. 5µl of the above stock diluted to 1ml using spray mix - to give 250fmoles/µl
4. 50:50 methanol: water (for cleaning syringe)
B. Perform a Blank run, this serves as a primary check:
1. Clean the syringe 3-4 times with 50:50 methanol: water.
2. Fill the syringe with spray mix and allow it to run for a few minutes.
3. Press Acquire (on the tune page) – give a filename and add details in the text box. Set the
mass range from 100 to 4000 m/z. Acquire around 200 scans
4. Allow the spray to stabilize. You will see some small masses (156.6676 and 213.2656corresponding to the spray mix solvent) in the spectrum. Check for response, ideally a
response of about 150 to 200 ions/scan is good. Record the spectrum – for reference
(Figure 2)
1.84e3
156.6676
%
100
213.2656
338.1360
282.7672
0
100
200
300
663.5245
664.5270
339.1455
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
m/z
Figure 2) Representative spectrum of a blank solvent (acetonitrile: water 50:50 (v:v) with 0.1%
formic acid)
C. Set the instrument to uncalibrated state:
Go to Calibration on the main menu and open Uncal.cal
D. Calibration with Horse Heart Myoglobin:
1. Fill the syringe with 250fmoles/µl of myoglobulin solution, and run the sample for a few
minutes.
2. Press Acquire (on the tune page) – give a filename for ex.120705_MYO_1 and add details in
the text box. Set the mass range from 500 to 2000 m/z.
3. Allow the spray to stabilize. You will see a charge series (typical of a protein-Figure2) in the
spectrum. Check for response, ideally a response of about 150 to 200 ions/scan is
good(excluding m/z 616.5276- corresponding to Heam)
UE511
500 fm of myoglobin in spray mix (1:1 mix ACN+H2O+0.1%FA)
05-Jul-201214:43:16
120705_MYO_3 177 (3.292) AM (Cen,4, 80.00, Ar,10000.0,0.00,1.00); Sm (SG, 2x10.00); Sb (2,40.00 ); Cm (150:200)
A22;771.5287
100
A24
707.3199
A20
848.5800
TOF MS ES+
4.31e5
A:
16951.50±0.18
A19
893.1901
A18
942.7568
A25
679.0671
A17
998.1547
%
A26
652.9880
A16
1060.4780
616.2143
A15
1131.1094
A14
1211.8263
A13
1304.9598
A28
606.4172
A12
1413.6233
A29
585.5357
312.9596
0
300
400
1312.5148
566.0504
500
600
700
800
900
1000
1100
1200
1300
A11
1542.0527
1421.7990
1400
1500
A10
1550.9885 1696.2312
1600
Figure 3) Representative spectrum showing the charge series of Horse Heart
Myoglobin at a concentration of 250 fmol/µl.
1700
1884.8702
1800
4. Stop the acquisition.
5. Re- Acquire – give a calibration filename for ex.120705_MYO_Cal_1.and add details in the
text box. Set the mass range from 500 to 2000 m/z.
6. Combine necessary amounts of scans to get a better spectra (Figure 4),
Figure 4): Screen shot of the Combine/Integrate spectra process menu of MassLynx software
7. Process the combined spectra, i.e.do a background subtraction, smooth and centroid using
Mass measure (Figure 5) from the process menu. Record the spectrum – for reference.
8. Save the spectrum – saves as AccMassxxx
9. Go to Calibration on the main menu
10. Here go to Calibrate
from File
Display Calibration Graphs – browse to
the file for ex.120705_MYO_Cal_1
go to history, you will find the saved AccMass file ,
click on it and say OK
11. A window opens with a report of number of matches and RMS residual value. To improve the
calibration, matches which are totally off can be removed by zooming and right clicking on the
1900
m/z
reference and data file uncalibrated (m/z match which was off will be removed).Go to
display default to see the improvement.
12. Then once satisfied with the number of matches and RMS residual value (at least 15-16 good
-1
matches out of 21 and RMS residual value of less than 4e ) - go to finished and accept
calibration, and do file save as- Save file with date-format YYMMDD_MYO_Cal.cal.
( Important never save over the Uncal.cal file , always use save as)
13. After calibration the mass of Myoglobin can be determined (deconvolution) by using the
spectrum after mass measure should be used. Go to Process
Components
Find
auto (Figure 6). Here give the mass range, for ex. 6000 to 20000 for Myoglobin and say find
First. It returns the mass of the protein.
Figure 5: Screen shot of the Mass Measure process menu of MassLynx software
Figure 6: The Deconvolution menu of MassLynx
14. Mass of the protein can also be determined by using maxEnt from the process menu, after
combining spectra.
15. Find the mass error in ppm (less than 10ppm is acceptable)
Expected mass of Horse heart Myoglobin is 16951.49 Da
III.
Determination of Intact mass of unknown sample
1. Requirements:
Obtain the following information from the user:
Amount of protein/compound in each sample, additive/contaminants/buffer composition of
sample, source of the sample, processing steps applied to purify the sample,
Protein sequence, Monoisotopic mass of the protein (can be calculated from the sequence) or
molecular formula of the compound, any other information such as SDS PAGE gel picture or
structure if available.
2. Vortex the sample for 30mins at 1500rpm, centrifuge at 5000rpm for 5 min
3. Supernatant to be taken for further dilutions using Spray mix.
4. Run a blank infusion with spray mix
5. Press Acquire (on the tune page) – give a filename and add details in the text box. Set the mass
range from 100 to 4000 m/z. Acquire around 200 scans.
6. Allow the spray to stabilize. You will see some small masses (156.6676 and 213.2656corresponding to the spray mix solvent) in the spectrum. Check for response, ideally a response
of about 150 to 200 ions/scan is good. Record the spectrum – for reference (Figure 1)
7. Sample analysis -Start with higher dilution 1:1000 ratio with Spray mix
8. Perform an initial survey scan with broader mass range to check the response of the compound
being injected. Press Acquire (on the tune page) – give a filename and add details in the text box.
Set the mass range from 100 to 4000 m/z. The mass range can be reduced if required after
seeing the response.
9. Allow the spray to stabilize. Check for response, ideally a response of about 150 to 200 ions/scan
is good.
10. If the response is low, dilutions like 1:200 or 1:50 ratio can be prepared accordingly.
11. Acquire with the dilution which gives good response – give a sample filename with user name
and sample ID (for example120705_NITYA_Sample_1) and add details in the text box.
12. Combine necessary amounts of scans to get a better spectra (Figure 4),
13. Process the combined spectra if necessary, i.e.do a background subtraction, smooth and centroid
using Mass measure (Figure 5) from the process menu. Record the spectrum – for reference.
14. The mass of the sample if it is a protein, can be determined (deconvolution) by using the
spectrum after mass measure should be used. Go to Process
Components
Find auto
(Figure 6). Here give the mass range depending on the expected mass of the sample, and say
find First. It returns the mass of the protein.
15. Mass of the protein can also be determined by using maxEnt from the process menu, after
combining spectra.
Best representative spectra with significant masses are provided to the user along with the
results.
Example: Determination of intact mass of β- casein(C6905- Sigma Aldrich, from bovine milk)
a)
b)
Figure 7) Direct infusion experiment of β-casein (C6905- Sigma Aldrich) with a concentration of
500fmol/µl in acetonitrile / water (1:1) with 0.1% formic acid. a) Total Ion chromatogram b) Single scan
spectra
Figure 8) Original spectrum obtained by combining 100 scans. Inlet for the m/z region 1400 – 1450
indicates two main components. Screenshot of used parameters
Figure 9) Processed spectrum obtained after background subtraction. Inlet for the m/z region 1400 –
1450. Screenshot of used parameters.
Figure 10) Processed spectrum obtained after smoothing. Inlet for the m/z region 1400 – 1450.
Screenshot of used parameters.
Figure 11) Processed spectrum obtained after performing centroidization processing. Inlet for the m/z
region 1400 – 1450. Screenshot of used parameters.
500fm beta casein in Spray mix (1:1 mix ACN+H2O+0.1%FA)
26-Jul-201216:49:01
A:
B:
26-Jul-201216:49:01
B17;1411.8181 A17;1414.1810
100
B17
24024.13±1.39
23983.74±0.65
A23;1045.5132
100
UE511
120726_BETA CASEIN _2 121 (2.253) AM (Cen,4, 80.00, Ar,10000.0,0.00,1.00); Sm (SG, 2x10.00); Sb (1,40.00 ); Cm (110:210)
TOF MS ES+
1.26e5
A24
1001.9899
A17
B17
1411.8181
A22
1092.9969
TOF MS ES+
1.22e5
A:
B:
24024.13±1.39
23983.74±0.65
1.26e5
A:
B:
24024.13±1.39
23983.74±0.65
A18
1335.6686
B18;1333.4241
A21
1144.9938
%
B24
1000.3210
B16
1500.0397
A19
1265.4198
B25
960.3467
A16
1502.5466
B19
1263.2935
1430.1826
1410.7786
1433.3215
1418.1567
1416.4646
1407.1277
1424.3477
1404.9028
B26
923.4496
0
1421.2871
1405
1410
1415
1420
1434.1931
1429.2866
1435.5857 1437.3466
1426.2332
1445.7789
1443.5360
1425
1430
1435
1440
1447.5656
1445
%
B15
1600.0775
B27
889.2890
A15
1714.5007
1602.7338
1523.0616
B28
857.5651
1846.7229
A14
1717.3584
1849.7990
1528.1837
A29
829.3992
2001.3844
1737.8524
1713.2202
0
800
2004.7087
1621.8368
900
1000
1100
1200
1300
1400
1500
1600
1700
1871.9204
1741.4902
1760.2448
1800
2027.6321
1897.2637
1900
2033.3821
2000
m/z
2100
Figure 12) Spectrum of β-Casein after assigning charge series using MassLynx deconvolution process
menu (Fig 5).
m/z
In our experimental approach we could identify two major genetic variants as described in [1]. Genetic
variant A1 was detected with experimental molecular mass 24092.1±1.4 and A2 was detected with
molecular mass of 2398.7±0.7.
References:
[1] J Proteome Res. 2009 March ; 8(3): 1347–1357. doi:10.1021/pr800720d
please refer to table 2 on page 26 :β-casein genetic variants,
Mass 24024.13 corresponds to variant A1
Mass 23983.74 corresponds to variant A2
Genetic variant B with a molecular mass of 24092.34 could not be identified in our experiment.