Application NoteProteomics

Absolute Quantification of Proteins in
Snake Venom
Using capLC-ICP-QQQ and online isotope dilution
analysis

Authors
Francisco Calderon-Celis,
Jorge Ruiz Encinar, AlfredoSanz-Medel
Department of Physicaland Analytical Chemistry,
University of Oviedo, Spain
Juan Jose Calvete
Instituto de Biomedicina deValencia, Consejo Superior deInvestigaciones Científicas(CSIC), Valencia, Spain

Introduction
Venoms are complex biological fluids that contain unique mixtures of peptides andproteins. Identifying and quantifying the composition of venoms is of increasing
scientific interest. It is especially important to characterize the toxins responsible forthe severe biological effects of the venom on humans. Better understanding of thesymptoms of envenoming would help with the development of effective therapies.
Also, venoms are being investigated as potential sources of new compounds in drugdevelopment.
In this study, an absolute quantification method suitable for the direct quantificationof intact proteins was applied to the analysis of the venom of the Mozambique
spitting cobra (Naja mossambica). This cobra is one of the most dangerous snakes
in Africa. The cobra’s venom is mainly toxic to cells (cytotoxic), causing swellingof the bite wound that may evolve into tissue necrosis and gangrene [1, 2]. The
cytotoxic components of the cobra venom have been identified mainly as membersof the three-finger toxin (3FTx) and phospholipase A2 (PLA2) protein families [3, 4].

The methodology was based on capillary liquid
chromatography (capLC) coupled to a triple quadrupole
ICP‑MS (ICP-QQQ). Absolute protein quantification wasachieved by measuring the sulfur heteroelement in theproteins, calibrated using online isotope dilution analysis(IDA). ICP-QQQ uses MS/MS mode to control reaction
chemistry in the collision/reaction cell (CRC), giving
consistent removal of spectral interferences using reactive
cell gases [5]. Efficient removal of spectral overlaps using
MS/MS allows access to multiple isotopes of biologicallyimportant elements such as iron, sulfur, and selenium.
ICP-QQQ enables the quantification of metalloproteins andpeptides using IDA, without the need for compound-specificcalibration standards [6, 7].
Most proteins (> 95%) contain sulfur from methionine andcysteine residues [8, 9], but sulfur determination is difficult
by conventional single-quadrupole ICP-MS. The challenges
are due to the element’s high ionization potential (10.4 eV)that leads to low sensitivity, and the occurrence of spectralinterferences from multiple polyatomic ions that overlap all
isotopes of sulfur. ICP-QQQ provides low backgrounds andhigh sensitivity, and removes multiple matrix interferencesusing MS/MS, freeing up the three most abundant S isotopes
for accurate measurement using IDA.
In this study, capLC-ICP-QQQ was used for the quantitative
analysis of intact proteins, isolated, and present in simple
mixtures. The method was also applied to the analysis andquantification of the major toxins comprising the venom
proteome of the Mozambique spitting cobra.

Instrumentation
Capillary LC separation was performed using an Agilent
1200 Series HPLC system fitted with a BIOShellTM A400 C4,3.4 μm, 150 mm x 0.3 mm reversed-phase column (SigmaAldrich, Germany) and autosampler. Chromatographic
column and post-column connections comprised Agilent
PEEK-coated fused silica capillaries 200 mm x 100 μm id (ICPand syringe connection) and 50 μm (column connection),and a 0.03" (0.8 mm) Agilent zero-dead volume T-connector.
Post-column flow was provided by a syringe pump system
kdScientific (Holliston, MA, USA). The column was heatedusing a Spark Holland oven (Mistral, The Netherlands) toimprove chromatographic efficiency.
Sulfur isotope measurements were carried out using anAgilent 8800 Triple Quadrupole ICP-MS (ICP-QQQ). The capLC
column was connected to the ICP-QQQ using the Agilent
capillary LC interface kit (Agilent product number G3680A).
The kit consists of a total consumption nebulizer with single
pass spray chamber. Enriched 34S was added to the LC eluate
solution post-column. Sulfur isotopes were measured byICP-QQQ using an oxygen mass-shift MS/MS method and theS content of the proteins was determined by IDA [10]. BOCMet-OH was used as the internal standard (IS) to correct for
any injection errors. capLC-ICP-QQQ operating conditions aregiven in Table 1.
Table 1. capLC-ICP-QQQ operating conditions.
ICP-QQQ
RF power (W)1550

Experimental

Sampling depth (mm)8.0

Carrier gas flow rate (L/min)

0.85

Reagents and samples

Makeup gas flow rate (L/min)

0.00

O2 cell gas flow rate (mL/min)

0.16

Data acquisition ion pairs,
Q1 (S+) » Q2 (SO+) mass (m/z)

32 → 48
34 → 50

Methionine and BOC-Met-OH (Sigma-Aldrich, Germany) wereused as standards. Bovine serum albumin (BSA), transferrin,β-casein, and cytochrome C (Sigma-Aldrich, Germany); andintact monoclonal antibody (mAb) Mass Check Standard(Waters, USA) were used as protein standards in the recovery
study. Other reagents included sulfur (1000 mg/L S) ICP
standard (Merck KGaA, Germany); solid isotopically enriched
34S (Isoflex USA); and sodium hydroxide (VWR ChemicalsBelgium). Lyophilized Naja mossambica venom was obtained
from the specialist venom supplier Latoxan S.A.S., France.
The venom was collected from a snake from Tanzania, andwas stored at -20 °C before use. All solutions were preparedin Milli-Q water (ChemLabor Millipore system, with 0.22 μmfilter, Millipak - Millipore). Mobile phase B was prepared inacetonitrile (ACN) Optima® LC/MS (Fischer Scientific, USA).
Formic acid (FA) was bought from Merck KGaA (Germany).

capLC
Chromatographic flow rate (μL/min)

4.5; 3.5*

Mobile phase A

H2O/0.2% FA

Mobile phase B

AcN/0.2% FA

Temperature (°C)
Chromatographic gradient80
Time range (Min)% mobile phase B

BSA and Intact mAb standards

021618226090

Naja mossambica sample

08104047

1.5
1.5103090* Conditions for measurement of venom sample

2

Results and Discussion
Absolute protein quantification
Absolute quantification of proteins was achieved through themeasurement of sulfur by capHPLC-ICP-QQQ, with postcolumn IDA. Two individual protein samples (BSA and mAb)
were spiked with BOC-Met-OH IS. Quantitative ID (masspurity) results for BSA, 95 ± 5% (n=3), compared well with thesample purity data provided by the manufacturer (≥ 98 %).
The ID mass purity result for Intact mAb was 77 ± 4% (n=3).
To confirm the accuracy and specificity of the ICP-MS/MS
analysis, digests of the two protein standards were quantified
using external calibration of sulfur. The results obtained byexternal calibration were 96 ± 1% for BSA and 79 ± 2% forIntact mAb. These results are in good agreement with the ID
mass purity results for BSA and Intact mAb shown above.

Quantitative analysis of snake venom proteome
The method was applied to the analysis and quantificationof the proteins present in a snake venom sample. Before
analysis, chromatographic recovery was calculated bymeasuring the sulfur mass of a sample eluting from thechromatographic column and comparing this to the sulfur
mass recorded directly by flow injection (FI). Chromatographic
recovery for a series of protein standards (cytochrome C,
β-casein, transferrin, BSA, Intact mAb) was higher than 98%.
For the Naja mossambica protein content, it was 99 ± 1%(n=3). The total sulfur mass content corresponded almost
exactly to the sum of the sulfur contained in the different
venom protein peaks detected via their sulfur heteroelement
content (Figure 1). The excellent recovery obtained for thecomplex sample shows that quantitative protein recoveries
for the chromatographic column are species (individualprotein) independent.

Parallel capLC-ESI-MS analysis was used to identify theproteins from their molecular weight, according to database
information (Table 2). By identifying the proteins, it waspossible to know the S-to-protein stoichiometry of each peak.
This information was then used to translate sulfur massinto individual protein quantities, in μmol protein per gramof venom sample. The quantified results are summarized inTable 2.
Table 2. Matching of the masses of protein peaks from Naja mossambica
venom to known protein families. Closest available protein species,
estimated exact mass, and calculated concentration are listed. Uncertainty
corresponds to one standard deviation (n=3).
Peak

FamilyClosesthomolog

Mol wt (Da)μmol protein/g
venom sample

13FTx

–

7064.2

1.99 ± 0.06

23FTx

~P29179

7417.40.471 ± 0.066

33FTx~P291797451.60.325 ± 0.040

43FTx~P014206892.41.10 ± 0.13

53FTx

~Q9W6W67786.4

< 0.1

63FTx

~P01452

7277.30.680 ± 0.050

73FTx~P01452

7306.30.668 ± 0.057

83FTx

–

7246.21.35 ± 0.10

93FTx

P255176832.45.09 ± 0.28103FTx

P014526704.319.0 ± 0.8

113FTx–6686.30.183 ± 0.035

123FTx–6829.30.220 ± 0.039

133FTx–6687.3< 0.114

PLA2P00604

13280.9

7.76 ± 0.32

153FTxP014706882.4

9.54 ± 0.27

163FTxP255176813.316.2 ± 0.4

173FTxP014676814.327.8 ± 0.8

183FTx~P014697046.45.13 ± 0.26

19PLA2P00604

13237.8

3.40 ± 0.14

20

PLA2P00002

13196.6

7.35 ± 0.33

21

PLA2–

13179.70.805 ± 0.04922Minor–

42000

0.102 ± 0.009

23Endonuclease

–

30000

0.619 ± 0.050

24SVMP

Q1074946700

0.165 ± 0.009

25

SVMP

Q1075046700

0.264 ± 0.013

26

SVMP

Q1075146700

0.097 ± 0.006

27

SVMP

Q1075246700

0.257 ± 0.006

Reprinted with permission from Anal. Chem., 2016, 88 (19), 9699–9706. Copyright 2016
American Chemical Society.

Figure 1. capLC-ICP-QQQ mass flow chromatogram of Naja mossambica
venom. All the venom protein species eluted between 20 and 50 min(S detection). The 27 sulfur-containing peaks are numbered—see Table 2 formore information.
Reprinted with permission from Anal. Chem., 2016, 88 (19), 9699–9706. Copyright 2016 American
Chemical Society.

3Conclusions

7.

P. De Raeve, J. Bianga, Fast and accurate absolutequantification of proteins and antibodies using Isotope
Dilution-Triple Quadrupole ICP-MS. Agilent publication,
2016, 5991-6118EN

Agilent ICP-QQQ instrumentation is especially suited for IDA
analysis of S as it uses MS/MS to remove multiple spectralinterferences from several S isotopes using an oxygen massshift method. By adding enriched 34S post-column and spiking
each sample with a generic S-containing internal standard,multiple S isotopes can be measured as SO+ product ions.
MS/MS ensures that each S isotope mass enters the CRC
in isolation, so no interfering product ions can be formed
from the other isotopes of S. This approach allows the sulfur
content of the proteins to be determined by IDA.

8.

M. Wind, A. Wegener, A. Eisenmenger, R. Keller, W.D.
Lehmann, Angewandte Chemie International, 2003,
42:3425–3427

9.

H. Zhang, W. Yan, R Aebersold, Current Opinions Chem.
Biol. 2004, 8 (1) 66–75

The capLC-ICP-QQQ method uses measurement of sulfur by
IDA to enable the absolute quantification of intact proteins
without the need for protein-specific standards.

The method was applied successfully to the quantificationand mass purity confirmation of protein standards. If
quantitative chromatographic recoveries can be assured,
it is even possible to quantify nonpure protein samples
using this method. The potential of the methodology for thequantification of intact proteins present in relatively complex
samples was demonstrated by analyzing 27 proteins in snakevenom.

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www.agilent.com/chem
For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc. 2020
Printed in the USA, March 25, 2020
5991-9016EN
DE.0575578704

10. F. Calderon-Celis, S. Diez-Fernandez, J. M. CostaFernandez, J. R. Encinar, J.J. Calvete, and A. Sanz-Medel,
Elemental Mass Spectrometry for Absolute Intact Protein
Quantification without Protein-Specific Standards:
Application to Snake Venomics, Anal. Chem., 2016, 88(19), 9699–9706.

More Information

The full results of the study were published in:Francisco Calderón-Celis et al., Elemental Mass Spectrometryfor Absolute Intact Protein Quantification without ProteinSpecific Standards: Application to Snake Venomics, Anal.
Chem., 2016, 88 (19), 9699–9706.