SRM vs HR-MS quantitative proteomics: matrix
effect and LOQ in serum
Dominique Baiwir
1,2, Gabriel Mazzucchelli
1, Nicolas Smargiasso
1and Edwin De Pauw
11
Laboratory of Mass Spectrometry, GIGA-R, chemistry department, CART, University of Liège,
2Proteomic Facility, GIGA, University of Liège
Overview
Introduc/on
Methods
Conclusions
Acknowledgements
-‐ 2D clean-‐up kit (GE) was applied according to the manufacturerrecommenda<ons for protein precipita<on
-‐ Protein diges<on was performed using trypsin, a@er reduc<on and alkyla<on -‐ Samples were dried under vacuum
-‐ Samples were recons<tuted in water 0.1% formic acid (v/v) and spiked with the 4 exogenous proteins
digest mix (MassPrep Diges<on Standard Mix2, Waters): Bovine Serum Albumine (BSA, P02769), Glycogen Phosphorylase B from rabbit (GPB, P00489), Enolase 1 (ENO, P00924) and Alcohol Deshydrogenase from
Saccharomyces cerevisiae (ADH, P00330)
-‐ Samples were spliXed in 2 and stored at -‐20°C un<l injec<on
-‐ Each samples was injected on triple quadrupole (TQ) and High Resolu<on – Mass Spectrometry (HR-‐MS) instruments
Mass spectrometry:
Results
UPLC:
Acquity I-‐Class (Waters): HSS T3 2.1*150 mm, 1.7 µm -‐ Column temperature: 40°C, Flow rate: 0.250 mL/min
-‐ 43 min linear gradient from 0% to 35% ACN 0.1% formic acid (v/v) (total run <me: 60min) -‐ Volume of injec<on: 9 µL
Experimental scheme
Comparison of SRM (Xevo TQ-‐S), t-‐MS² and t-‐SIM (Q Exac/ve)
2-‐D clean-‐up kit
Trypsin diges<on
Spike of
4 proteins digest
Triple quadrupole
(SRM)
HR-‐MS
t-‐MS² / t-‐SIM
HR-‐MS
t-‐MS²:
Obvious choice of the right
peak
HR-‐MS spectra (t-‐MS²) for VNQIGTLSESIK with 42 µg matrix
Serum is one of the gold standards matrix for diagnosis or prognosis
purposes
Most of proteins that are indicators of disease state or progression lies
in the low serum concentra<on range
Serum is very complex due to :
• Large protein concentra<on dynamic range
• High number of different proteins present
depending on the physiological state → Matrix effects ? LOD / LOQ ?
GOAL OF THIS STUDY:
compare triple quadripole versus HR-‐MS technologies
-‐ matrix effects
-‐ LOQ HR-‐MS (Orbitrap):
Q Exac<ve (Thermo)
Comparison of results obtained with the 2
insruments Triple quadrupole: Xevo TQ-‐S (Waters)
SRM
• Interference on transi<on 644.9>834.5: (M+2H)2+ > y8+ • Chromatogram 42µg of matrix • Area increaseswhen amount of matrix increases
• Not suitable for
quan<fica<on
• 2 other transi<ons are
rather constant when matrix amount is
increased
• Suitable for
quan<fica<on
•
In triple quadrupole, matrix interferences are observed on several transi/ons ( predicted as the most intense)
•
Transi/ons dependent, not predictable
•
t-‐MS² with HR-‐MS is the less sens//ve to the matrix effect of serum
•
All pep/des are detected with equivalent area whatever the matrix amount
•
Use of HR-‐MS in t-‐MS² mode for transi/ons selec/on for triple quadrupole
•
Most intense fragments are for the majority (~66%) the highest ones obtained using Xevo TQ-‐S
• Targeted MS² allows to easily iden<fy the chromatographic peak corresponding to the target pep<de
• Provides also informa<on on interfering compounds
• It allows to easily select the most intense fragments and to check for matrix interference
The F.N.R.S., European Union (FEDER) and Walloon Region contributed to the contributed the mass spectrometry laboratory and the GIGA Proteomics Facility fundings.
Q Exac<ve (Thermo):
-‐ Targeted MS²: resolu<on: 35000, AGC target: 5 105, max accu <me : 125 ms, isola<on window:
3 m/z, isola<on offset: 0.5 m/z.
-‐ Targeted SIM: resolu<on: 140000, AGC target: 2 105, max accu <me: 125 ms, isola<on window:
3 m/z, isola<on offset: 0.5 m/z, MSX count (mul<plex): 4. Xevo TQ-‐S (Waters): Selected Rea<on Monitoring (SRM)
-‐ Source parameters: capillary 2.0 kV, cone 35 V, source offset: 50 V, Desolvata<on gas flow: 1000 L/h -‐ LM 1 / 2 Resolu<on: 2.8 / 2.8, HM 1 / 2 Resolu<on: 14.9 / 14.8
-‐ 3 transi<ons (y ions) per pep<de
-‐ 3 <me windows with 24, 15 and 24 transi<ons : auto-‐dwell <me (12 points per peak , FWHH of 8 s)
Selected pep/des
Pep<des were selected according the following criteria: -‐ Already observed in discovery method using MPDS
Mix
-‐ Absence of the pep<de sequence in human proteome (BLAST)
-‐ Not possible for GPB pep<des -‐ Length between 4 and 40 amino acids
-‐ No cysteine, no methionine in pep<de sequence -‐ No poten<al glycosyla<on site (NXT, NXS)
-‐ No miss cleavage (no RP/KP for cleavage site to avoid par<al diges<on)
Pep<de VNQIGTLSESIK of Enolase (465 fmoles – 22 ng)
SRM
t-‐MS²
t-‐SIM
RT:20.65 - 24.69 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 Time (min) 0 10 20 30 40 50 60 70 80 90 100 R e la tive A b u n d a n ce RT: 22.37 21.20 22.77 20.72 20.80 20.96 21.45 21.91 22.16 23.71 23.68 23.96 23.29 24.4424.55 23.06 24.07 NL: 4.61E6 TIC F: FTMS + p ESI Full ms2 644.86@hcd25.00 [89.00-1335.00] MS 140528-70ug-360fmol-tMS2-01 Interfering pep<de140528-70ug-360fmol-tMS2-01 #2074-2125RT:21.13-21.25AV:6NL:2.09E5
F:FTMS + p ESI Full ms2 644.86@hcd25.00 [89.00-1335.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z 0 10 20 30 40 50 60 70 80 90 100 R el at ive A bu nd an ce 603.86 456.26 337.15 618.86 833.45 670.39 147.11 555.33 872.54 238.12 408.22 246.18 129.10 183.11 546.82 932.52 359.26 771.50 878.46 488.31 682.32 1000.60 1109.66 595.35 815.44 300.15 1128.69 1288.27
140528-70ug-360fmol-tMS2-01 #2470-2544RT:22.31-22.51AV:13NL:7.45E4
F:FTMS + p ESI Full ms2 644.86@hcd25.00 [89.00-1335.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z 0 10 20 30 40 50 60 70 80 90 100 R e la tive A b u n d a n ce 834.46 342.18 101.07 644.36 214.12 947.54 120.08 243.11 563.30 393.21 529.30 147.11 676.39 197.09 325.15 456.26 777.43816.45 1075.60 476.27 260.20 356.19 626.35 909.47 852.45 1140.55 996.50 688.35 1183.58 1261.79 Expected pep<de 140528-70ug-360fmol-tMS2-01 #2602-2679RT:22.70-22.91AV:11NL:5.81E5
F:FTMS + p ESI Full ms2 644.86@hcd25.00 [89.00-1335.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z 0 10 20 30 40 50 60 70 80 90 100 R e la tive A b u n d a n ce 644.37 175.12 243.13 357.18402.25 627.34 158.09 442.26 514.26 722.38 833.42 915.46 1006.53 691.34 1063.34 1176.41 1318.68 793.78
Single charged interference
0.6µg matrix 42µg matrix min 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 % 0 100 F1:MRM of 24 channels,ES+ 644.9 > 676.4 140430-MS_70ug_360fmolesADH Smooth(Mn,1x2)
Matrix effect analysis - 70 ug Serum - 360 fmoles ADH MPDSMIX2
1.105e+005 ENO - VNQIGTLSESIK 22.60 9557.75 23.95 min % 0 100 F1:MRM of 24 channels,ES+ 644.9 > 777.4 140430-MS_70ug_360fmolesADH Smooth(Mn,1x2)
Matrix effect analysis - 70 ug Serum - 360 fmoles ADH MPDSMIX2
3.916e+004 ENO - VNQIGTLSESIK 22.60 3391.95 min % 0 100 F1:MRM of 24 channels,ES+ 644.9 > 834.5 140430-MS_70ug_360fmolesADH Smooth(Mn,1x2)
Matrix effect analysis - 70 ug Serum - 360 fmoles ADH MPDSMIX2
4.971e+005 ENO - VNQIGTLSESIK 22.60 42366.17 21.11 21.33 RT:20.65 - 24.69 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 Time (min) 0 10 20 30 40 50 60 70 80 90 100 R el at ive A bu nd an ce 22.37 20.8220.89 21.01 21.16 21.25 21.4121.49 21.64 23.96 21.76 21.8722.06 23.06 22.85 22.62 NL: 2.39E5 m/z= 834.40-834.51 F: FTMS + p ESI Full ms2 644.86@hcd25.00 [89.00-1335.00] MS 140528-70ug-360fmol-tMS2-01 RT:20.65 - 24.69 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 Time (min) 0 10 20 30 40 50 60 70 80 90 100 R e la ti ve A b u n d a n ce 22.37 23.95 23.97 22.48 NL: 3.24E4 m/z= 675.90-676.89 F: FTMS + p ESI Full ms2 644.86@hcd25.00 [89.00-1335.00] MS 140528-70ug-360fmol-tMS2-01
LOD of SRM (Xevo TQ-‐S) and t-‐MS² (Q Exac/ve)
Pep<de of BSA (58 fmoles – 4 ng)
SRM
t-‐MS²
• In SRM
• the 3rd transi<on (y7) is not detected
• Ion ra<o (y5/y6) is OK in all
samples except 2 (60 µg and 90 µg matrix)
• In targeted MS²
• Detec<on in all samples except 2( ) (60 µg and 100 µg matrix) • Ion ra<o not constant through
samples series
~ 50 fmoles: LOQ in SRM, LOD in t-‐MS²
RT:20.90 - 24.70 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 Time (min) 0 10 20 30 40 50 60 70 80 90 100 R e la ti ve A b u n d a n ce 22.40 22.43 22.38 22.47 22.48 22.36 22.51 24.66 21.2821.31 22.8222.85 23.10 23.99 21.23 21.81 22.33 24.04 20.99 21.65 21.9222.13 22.77 23.1423.35 23.7423.97 24.2724.59 NL: 1.18E6 m/z= 643.86-646.86 MS 140528-1ug- 360fmol-tSIM-01 RT:20.62 - 24.52 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 Time (min) 0 10 20 30 40 50 60 70 80 90 100 R e la ti ve A b u n d a n ce 21.22 21.18 22.76 22.36 21.24 22.39 22.78 22.74 22.41 20.67 21.27 20.79 21.15 22.81 20.93 21.30 22.43 22.45 21.3421.54 22.33 22.83 21.56 21.7822.1522.18 22.4922.72 22.8522.87 23.68 23.7023.9523.97 23.65 24.00 24.09 24.45 22.52 22.9023.07 23.29 23.33 23.74 NL: 2.14E6 m/z= 643.86-646.86 MS 140528- 70ug- 360fmol-tSIM-01 0.6µg matrix 42µg matrix RT:20.65 - 24.69 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 Time (min) 0 10 20 30 40 50 60 70 80 90 100 R e la ti ve A b u n d a n ce RT: 22.37 21.20 22.77 20.72 20.80 20.96 21.45 21.91 22.16 23.71 23.68 23.96 23.29 24.4424.55 23.06 24.07 NL: 4.61E6 TIC F: FTMS + p ESI Full ms2 644.86@hcd25.00 [89.00-1335.00] MS 140528-70ug-360fmol-tMS2-01 RT:20.65 - 24.65 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 Time (min) 0 10 20 30 40 50 60 70 80 90 100 R e la ti ve A b u n d a n ce RT: 22.50 20.8420.94 21.34 21.6621.78 22.10 22.91 23.1623.25 23.56 24.0424.07 24.37 24.50 NL: 2.47E6 TIC F: FTMS + p ESI Full ms2 644.86@hcd25.00 [89.00-1335.00] MS 140528-1ug-360fmol-tMS2-01 0.6µg matrix 42µg matrix
All the selected pep/des give the same conclusion (except for SIM, for which results are very variable): matrix effect is very dependent of the
selected transi/on and does not seem to be predictable (not related to pep/de length, hydrophobicity,…)
t-‐MS²
• Interference on
834.4562 (y8+)
• Can be avoided
with the
fragment exact mass
• Reliable
quan<fica<on of the pep<de when the matrix amount increases
• No matrix effect
observed!
140528-70ug-360fmol-tMS2-01 #2466-2551RT:22.31-22.53AV:14NL:6.94E4
F:FTMS + p ESI Full ms2 644.86@hcd25.00 [89.00-1335.00] 833.0 833.5 834.0 834.5 835.0 835.5 836.0 836.5 837.0 837.5 838.0 m/z 0 10 20 30 40 50 60 70 80 90 100 R e la ti ve A b u n d a n ce 834.46 835.46 836.46 833.45 837.47 834.92
Interference (problem in SRM ! )
