Elemental Mass Spectrometry for Absolute Intact Protein Quantification

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ELEMENTAL MASS SPECTROMETRY FOR ABSOLUTE INTACT PROTEIN QUANTIFICATION WITHOUT PROTEINSPECIFIC STANDARDS: APPLICATION TO SNAKE VENOMICS. Francisco Calderon-Celis, Silvia Diez-Fernandez, Jose Manuel CostaFernandez, Jorge Ruiz Encinar, Juan Jose Calvete, and Alfredo Sanz-Medel Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02585 • Publication Date (Web): 03 Sep 2016 Downloaded from http://pubs.acs.org on September 4, 2016

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Analytical Chemistry

1

ELEMENTAL MASS SPECTROMETRY FOR ABSOLUTE INTACT PROTEIN

2

QUANTIFICATION

3

APPLICATION TO SNAKE VENOMICS.

WITHOUT

PROTEIN-SPECIFIC

STANDARDS:

4 5

AUTHORS:

6

Francisco Calderón-Celis1*, Silvia Diez-Fernández1*, José Manuel Costa-Fernández1, Jorge Ruiz

7

Encinar1+, Juan J. Calvete2,$, Alfredo Sanz-Medel1+

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1. Department of Physical and Analytical Chemistry, University of Oviedo, Julián Clavería 8,

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33006 Oviedo, Spain

11

2. Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas

12

(CSIC), Jaume Roig 11, 46010 Valencia, Spain

13

*These authors contributed equally to this work

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+ Corresponding authors for ICP-MS analysis. E-mails: [email protected] and

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[email protected]

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$ Corresponding author for questions of snake venomics. E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT:

2

Absolute protein quantification methods based on molecular mass spectrometry usually require

3

stable isotope-labeled analogous standards for each target protein or peptide under study, which in

4

turn must be certified using natural standards. In this work, we report a direct and accurate

5

methodology based on capLC-ICP-QQQ and on-line isotope dilution analysis for the absolute and

6

sensitive quantification of intact proteins. The combination of the post-column addition of 34S and a

7

generic S-containing internal standard spiked to the sample provides full compound independent

8

detector response and thus protein quantification without the need for specific standards.

9

Quantitative recoveries, using a chromatographic core-shell C4 column for the various protein

10

species assayed were obtained (96-100%). Thus, the proposed strategy enables the accurate

11

quantification of proteins even if no specific standards are available for them. In addition, to the best

12

of our knowledge, we obtained the lowest detection limits reported in the quantitative analysis of

13

intact proteins by direct measurement of sulfur with ICP-MS (358 fmol) and protein (ranging from 7-

14

15 fmol depending on the assayed protein). The quantitative results for individual and simple

15

mixtures of model proteins were statistically indistinguishable from the manufacturer’s values.

16

Finally, the suitability of the strategy for real sample analysis (including quantitative protein

17

recovery from the column) was illustrated for the individual absolute quantification of the proteins

18

and whole protein content in a venom sample. Parallel capLC-ESI-QTOF analysis was employed to

19

identify the proteins, a prerequisite to translate the mass of quantified S for each chromatographic

20

peak into individual protein mass.

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KEYWORDS:

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HPLC-ICP-MS, ICP-QQQ, absolute protein quantification, isotope dilution analysis, quantitative venomics

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ABSTRACT GRAPHIC:

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INTRODUCTION

2

Mass spectrometry (MS) has become a most powerful analytical technique for protein analysis1.

3

During the last 20 years MS evolved from mostly qualitative protein expression and PTM profiling

4

work into more focused quantitative studies2. In fact, the biomedical importance of proteins levels

5

determination (as an indicator of pathological conditions, health bio-markers and/or potential

6

therapeutic targets3) has placed quantitative proteomics at the focus of research in biology and

7

medicine4–7. It has a great potential for understanding biological functions, for the diagnosis of

8

diseases, and to support the development of new drugs8,9. In this sense, knowledge of absolute

9

quantities would allow to accurately gather information on protein levels, dynamics of post-

10

translational modifications (PTMs), or the changes in subunit stoichiometry of a protein complex in

11

response to environmental changes2,10. Absolute quantitative measurements are therefore more and

12

more demanded for the full characterization of the components of biological systems and for the

13

monitoring of their evolution during a pathological process.

14

Mass spectrometry proteomics is clearly turning quantitative7 while “absolute” quantification of

15

proteins, still in its infancy, is a clear aim nowadays11. The most extended absolute quantification

16

approaches involve the use of a synthetic stable isotopically-labeled (SIL) fragment (or the whole

17

molecule) of the protein of interest and selected or multiple reaction monitoring12–16. The fact that a

18

specific standard is required for each protein/peptide target limits the throughput of MRM

19

experiments to the measure of a restricted number of proteins simultaneously. The most tedious,

20

essential steps though, in a MRM experiment are the bioinformatic-guided prediction of the better

21

transitions for each target protein17, and the synthesis and accurate quantification (usually by amino

22

acid analysis) of pure SIL standards18, the latter being expensive, time-consuming, and requiring

23

significant amounts of the starting material19,20. Thus, while targeted protein quantification may be

24

useful in many clinical proteomics applications, there is still a pressing need for developing simpler

25

non-targeted analytical procedures for absolute quantification of proteins and peptides. In this regard,

26

the capability of inductively coupled plasma mass spectrometry (ICP-MS) for robust, accurate and

27

precise absolute isotope abundance measurements of heteroatoms (e.g. any atom different from C, O,

28

H or N), has opened the door to using isotope dilution analysis (IDA) to achieve absolute

29

determination of proteins via “heteroatom-tagged proteomics”21. The main advantage of such

30

elemental MS approach is that only one certified generic standard (i.e. containing an isotopically

31

labeled heteroatom of the naturally present element measured) is required to obtain absolute



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1

quantitative data for each chromatographically separated peptide or protein in the analyzed

2

sample21,22.

3

Nowadays ICP-MS based approaches in proteomics are extensively applied not only to the analysis

4

of metallic elements present in metalloproteins but to the determination of other heteroatoms

5

naturally present in proteins (e.g. sulfur, phosphorous or selenium) as well. In this sense, sulfur is a

6

most promising elemental target since the sulfur-containing amino acids methionine and cysteine are

7

statistically present in almost all proteins. Unfortunately, ICP-MS-based methodologies have been

8

traditionally limited by poor sensitivity and selectivity for non-metals Recently, however, the

9

introduction of the tandem MS concept (triple quadrupole QQQ typically used in molecular MS) for

10

ICP-MS enabled LODs in the low fmol range for S-containing peptides23. So far, however, such tool

11

has not yet been applied to the direct quantification of proteins, mostly due to frequent column

12

recovery limitations.

13

On the other hand, N. mossambica, considered one of the most dangerous snakes in Africa, is the

14

most common venomous snake of the savanna regions of tropical and subtropical Africa, ranging as

15

far south as Durban in the South African province of KwaZulu-Natal, northwards through the

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Mpumalanga Province Lowveld region, south-eastern Tanzania, Pemba Island in the Zanzibar

17

Archipelago, and west to southern Angola and northern Namibia24,25. The venom of the Mozambique

18

spitting cobra is predominantly cytotoxic, causing progressive and painful swelling of the bite

19

wound, with blistering and bruising, that may evolve into tissue necrosis and gangrene26,27. The

20

cytotoxic components of the Mozambique spitting cobra venom have been identified as members of

21

the three-finger toxin (3FTx) and phospholipase A2 (PLA2) protein families28,29. Detailed knowledge

22

of the composition and abundance of the venom proteome of medically important snakes is sine qua

23

non to understanding the clinical symptoms caused by their bites.

24

Here, we report the application of a hybrid methodology, based on capillary liquid chromatography

25

(LC) with new core-shell technology able to provide quantitative recoveries, coupled to ICP-QQQ,

26

for the quantitative analysis of intact proteins, both isolated or present in simple mixtures. The

27

validity of the approach for real sample analysis has been also evaluated by quantifying the major

28

toxins comprising the venom proteome of the Mozambique spitting cobra, Naja mossambica.

29

Our results indicate that elemental MS, via ICP-MS (QQQ) could offer a novel avenue to investigate

30

venom toxins, paving the way to new quantitative insights into integrative venomics30–32.

31

EXPERIMENTAL SECTION


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Reagents and materials

2

Pure standards Methionine and BOC-Met-OH (BOC) were purchased from Sigma-Aldrich

3

(Steinheim, Germany). Sulfur (S) ICP standard (1000 mg/L S) was purchased from Merck KGaA

4

(Darmstadt, Germany). Solid isotopically-enriched

5

Francisco, CA, USA). Sodium hydroxide was purchased from VWR Chemicals (Lieven, Belgium).

6

Bovine serum albumin (BSA), transferrin, β-casein and cytochrome C were from Sigma-Aldrich

7

(Steinheim, Germany) and Intact monoclonal antibody (mAb) Mass Check Standard was from

8

Waters (Milford, MA, USA). Venom from N. mossambica from Tanzania was purchased from

9

Latoxan S.A.S. (Valence, France) in lyophilized form and was stored at -20 °C until used.

34

S was purchased from Isoflex USA (San

10

All solutions were prepared in MilliQ water, obtained from ChemLabor Millipore system, with 0.22

11

µm filter (Millipak - Millipore). Mobile phase B was prepared in Acetonitrile (AcN) Optima®

12

LC/MS, purchased from Fischer Scientific (USA). Formic acid was purchased from Merck KGaA

13

(Germany).

14

Instrumentation

15

µHPLC separation was performed in an Agilent 1200 Series (Agilent Technologies, Waldbronn,

16

Germany) HPLC system equipped with a BIOShellTM A400 C4, 3.4 µm, 150 mm x 0.3 mm

17

reversed-phase micro HPLC column (Sigma) and autosampler. Chromatographic connections

18

(column connections and post-column configuration) were done with Fused Silica peeks (Agilent,

19

Germany) of 200 mm length and internal diameter of 100 µm (ICP and syringe connection) and 50

20

µm (column connection), and a 1/32” Agilent zero-dead volume T-connector. Post-column flow was

21

provided by syringe pump system kdScientific (Holliston, MA, USA). Spark Holland oven (Mistral,

22

The Netherlands) was employed as column heating system to improve chromatographic efficiency.

23

The complete configuration is described in Figure S1.

24

ICP-MS system consisted on a Triple Quadrupole ICP-MS, ICP-QQQ (Agilent 8800, Tokyo, Japan).

25

Capillary LC interface Total Consumption nebulizer (Agilent) was used as interface between the

26

µHPLC and the ICP systems. ESI-QToF MS was performed in a Bruker Daltonics (Bremen,

27

Germany) Impact II instrument. Experimental conditions are indicated in Table S1.

28

Procedures

29

Preparation and certification of protein samples and standards



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Solutions of S-containing internal standard and protein standards mAb were prepared by dissolving

2

the corresponding amount of compound or protein in mobile phase A. Protein mass purity was

3

assessed measuring the sulfur content of the digested protein samples by external calibration.

4

To synthesize the

5

sodium hydroxide solution at 80°C. Polysulfide was completely oxidized to sulfate by dropwise

6

addition of hydrogen peroxide solution (10% (v/v)) following the protocol described elsewhere19.

7

The solution was finally diluted with MilliQ water. The 34S-spike was fully characterized by isotope

8

dilution analysis, as described in Supplementary Information (see Equations S1-S3). Abundance of

9

32

34

S-enriched isotopic spike solution, elemental

34

S was dissolved in 30% (w/w)

S, 33S and 34S isotopes obtained for the spike solution were 3.01 ± 0.03%, 1.24 ± 0.02 % and 95.75

10

± 0.13 % respectively. Sulfur concentration in the initial spike solution was 10.46 ± 0.08 µg/g S.

11

Isotope dilution analysis

12

For the quantification of protein by isotope dilution analysis, both

13

monitored by ICP-MS. The signal ratio of both isotopes implies firstly the correction of signal

14

sensitivity variations, as 34S (added by means of a post-column flow) acts as internal standard23. This

15

signal enhancement (of almost five fold) along the chromatographic gradient has been already

16

described33,34. This justifies the use of post-column isotope dilution so as to get the response factor

17

for sulfur to remain constant along all the chromatogram as can be clearly seen in Figure S2.

18

The experimentally obtained 32S/34S ratio can be employed in the mass flow equation35, as described

19

below. An additional advantage of using an internal standard is that the relatively high complexity of

20

the isotope-dilution calculations is reduced to a minimum36,37. Briefly, from the mass flow equation

21

(Equation 1), the mass of S for the internal standard (mstd, known) and the mass of S for the species

22

of interest (ms, unknown) are related to the areas under the corresponding peaks (Astd and As,

23

respectively), as stated in Equations 2 and 3, which must be integrated in the same mass-flow

24

chromatogram. MFs = MFsp

25

34 32 / 34 − Rsp32 / 34 Aws Asp Rb Awsp As32 1 − Rb32 / 34 Rs32 / 34

Equation 1

26

mstd ( ng S ) = ∫ tt12 MFstd (t ) ⋅ dt = peak area = Astd

Equation 2

27

ms ( ng S ) = ∫ tt12 MFs (t ) ⋅ dt = peak area = As

Equation 3


32

S and

34

S isotope signals were

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The response factor obtained for the internal standard (mstd /Astd) can then be used to calculate

2

accurately and precisely the mass of S present in each chromatographic peak, simply by assuming an

3

arbitrary tracer mass flow (Equation 4). Of course, such a simplification requires that the tracer mass

4

flow should be completely stable along the chromatogram (a condition fulfilled in our system, as

5

stated above).

6

mS = mstd

As Astd

Equation 4

RESULTS AND DISCUSSION

7 8

Generic absolute quantification approach

9

Column recovery evaluation

10

It is well established that the protein mass eluted from a chromatographic column is usually not

11

quantitative38. This is especially relevant if quantification is accomplished directly from the

12

chromatographic peak without any SIL standard, as the column recovery factor will have a direct

13

influence on the calculated protein concentration. Therefore, it is necessary either to calculate

14

column recovery and assure its reproducibility along time, or to demonstrate that chromatographic

15

conditions assure quantitative recovery from the column for any protein species. Of course, the first

16

option demands for specific standards. In contrast, the fulfilment of the latter premise hence implies

17

that recovery does not have to be calculated for the individual protein species under analysis,

18

enabling a more generic applicability of the methodology. In order to demonstrate here that generic

19

quantitative column recovery was achieved, chromatographic and flow injection analyses (FI) were

20

compared for individual protein standards. Thus, by integrating the observed mass flow peaks and

21

comparing the sulfur masses obtained in both cases, the percentage of protein mass that is eluting

22

from the chromatographic column (for a set of proteins of different nature covering a wide range of

23

molecular weights from 12 to 145 KDa) was determined. The use of IDA in both cases ensures here

24

the feasibility of this procedure (the sulfur response was linear and thus independent of elution time

25

and species types). Consequently, the integrated peak areas obtained both in chromatography and in

26

flow injection analysis were compared. As an example, Figure S3 shows the FI and the

27

chromatographic peaks obtained for BSA protein, while the results listed in Table 1 show that the

28

chromatographic recovery is quantitative for all the six proteins tested. As a result, such recovery

29

calculation is no longer needed when analyzing protein samples under these experimental conditions.

30

This aspect is especially important for proteins for which no standard is available.



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1

Absolute protein quantification

2

The absolute protein quantification methodology proposed in this work is based on the use of generic

3

internal standards to support IDA for the quantification of the desired proteins. Although IDA

4

provides absolute quantification of the heteroelement, by spiking a S-containing internal standard to

5

the sample we can correct any errors during the injection (typical injection volumes 1-2 µl).

6

Moreover, there will be no need for correcting mass bias anymore as it affects in the same way the

7

internal standard and the analyte. Unlike molecular MS-based absolute quantification approaches,

8

the sulfur-containing standard does not need to be specific for each protein. The election of this

9

standard is based solely in chromatographic criteria (resolution, retention and separation from the

10

corresponding protein peak). In our case, BOC-Met was used as the internal standard (I.S.).

11

BSA and mAb samples were spiked with such I.S. and analyzed by capHPLC-ICP-QQQ, using the

12

post-column IDA approach (Figure 1A and 1B). The sulfur concentration in the proteins was

13

calculated from the corresponding peak areas ratio and I.S. concentration obtained by IDA. In this

14

way, we ensure that the response factor for sulfur remains constant along the whole chromatogram

15

gradient. Therefore, an accurate quantification by peak areas ratio from the mass flow chromatogram

16

of each protein species and the internal standard (as indicated by the equations displayed in the

17

experimental section) is secured. In any case, it is imperative to know the sequence of the desired

18

protein (and hence its number of sulfurs atoms per protein molecule), in order to be able to transform

19

sulfur concentration into protein concentration. In our case, model protein standards of known

20

sequences were tested.

21

The calculated BSA mass purity value of 95 ± 5% (n=3) compared well with the sample purity

22

provided by manufacturer (≥ 98 %). Additionally, in order to validate further the proposed

23

methodology, the protein standards used were subjected to acidic digestion then and quantified by an

24

external calibration of sulfur (of course, this complementary approach is only valid for highly pure

25

standards that do not contain any other sulfur containing compounds or impurities). Then, the

26

experimental mass purity figures gathered for BSA (and Intact mAb, of 77 ± 4% (n=3)) were in

27

perfect agreement with the similar measurement results obtained by external calibration: 96 ± 1% for

28

BSA and 79 ± 2% for Intact mAb.

29

In brief, ICP-MS based accurate absolute quantification of target proteins by using a simple, cheap

30

and generic standard and a direct chromatographic analysis is demonstrated. Moreover, in principle,

31

the sample does not need to be previously purified, since impurities, such as small inorganic species,

32

can be previously separated in the chromatographic step (see Figure 1). So, as long as species are


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1

separated and peaks well resolved, absolute quantification can be accomplished in one

2

chromatographic run, just requiring one generic sulfur-containing standard. Of course, peak purity

3

could be easily checked by parallel capLC-ESI-MS and assessment of the MS spectra at the elution

4

time of the target peak.

5

The applicability of the approach to a simple mixture of protein was carried out as well. To this end,

6

a mixture of three of the studied proteins (cytochrome C, transferrin and BSA) with BOC-Met as I.S.

7

was assayed (Figure 1C). Results were compared with expected mass purity (given by

8

manufacturers) and determined by alternative methods (Biuret for transferrin, UV-spectroscopy for

9

cytochrome C and agarose electrophoresis for BSA). The comparison of mass purity values provided

10

by the manufacturer and those determined by our methodology is shown in Table 2. As can be seen,

11

quantitative results observed are statistically indistinguishable for those expected, which validates

12

further the proposed approach.

13

Analytical characteristics of the proposed approach

14

Since protein quantification is achieved by means of relating the integrated peak areas of both the

15

internal standard and the protein of interest, the chromatographic characteristics of the analysis are

16

particularly relevant. Peak purity is a requisite as ICP-MS-based detection cannot distinguish

17

between sulfur coming from one protein or another species in a mixture. Well resolved peaks are

18

therefore of utmost importance in order to achieve accurate integration of the whole chromatographic

19

peak (and so reliable quantification). In this sense, the use of a core-shell column has proved to

20

provide the needed performance, such as chromatographic efficiency for the different analyzed

21

species (10000 – 35000), peak widths in the range of 15-36 s, and asymmetry factor between 1.1 and

22

1.2 (that is, the peaks showed negligible tailing). Regarding separation of the peaks, chromatographic

23

resolutions between 3.4 and 4.6 were obtained for the protein mixture, indicating that the peaks were

24

well (baseline) separated. The use of comparatively high temperature (80ºC) during the

25

chromatographic separation was important here (enhanced efficiency with higher temperature of the

26

column for protein analysis has been already reported39).

27

Analytical potential of sulfur measurement for the quantitative analysis of proteins and peptides has

28

already been pinpointed40. In addition, recent studies using a triple quadrupole ICP-MS (QQQ)

29

proved significant LOD enhancement for the quantitative analysis of peptides through their sulfur

30

atoms measurement23 (down to 11 fmol of peptide). However, to the best of our knowledge, so far

31

direct ICP-MS-based quantification of intact proteins by means of measuring elemental sulfur, either

32

by calibration41,42 or by isotope dilution19,43,44, provided LODs in the range of 1.2-300 pmol S. In this



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work, calculated LODs for the quantification of sulfur (calculated considering the standard deviation

2

of the baseline and the net peak height) were 108 fmol for the I.S. and 280-590 fmol for the protein

3

standards.

4

Such LODs are thus higher than those reported by Diez et al23 (11 fmol) for S-containing peptides

5

using also the ICP-QQQ. Since LOD calculation is based on net peak height, protein

6

chromatographic peaks are wider (15-36s) than typical peptide peaks (9s). Therefore, LODs are

7

worse here. Besides, the use of a post-column flow of enriched-in-96%

8

addition of a sulfur-containing solution, where about 3% out of the sulfur content will be in form of

9

32

34

S implies the constant

S, (see Experimental Section), thereby increasing the baseline and consequently worsening the

10

LODs. Nevertheless, unlike peptides, which usually have just one sulfur atom, tens of sulfur atoms

11

can be found in proteins (e.g. 39 S/BSA and 52 S/Intact mAb). Consequently, whereas in the case of

12

peptides S LOD is equivalent to the observed species LOD, when LODs for sulfur translate into

13

protein LODs result in 7-15 fmol values (quite similar to the values obtained for peptidic species in

14

reference (20)).

15

Quantitative analysis of Naja mossambica venom proteome

16

The application of omics methodologies to the study of venoms has grown steadily in recent years.

17

Locus-resolved top-down venom proteomics is rapidly making its way in the context of integrative

18

venomics. However, the absolute quantification of the venom components, an essential data for

19

integrating structural and functional venomics, is still a pending issue. The most commonly

20

employed method to quantify the individual proteins or the protein family abundance in a venom

21

involve monitoring the reversed-phase column eluate at the absorbance wavelength of the peptide

22

bond, 215-220 nm30,32. According to the Lambert–Beer law (A = εlc, where A = absorbance; ε molar

23

absorption coefficient and c = concentration [M]. The protein percentages determined in the eluate

24

correspond to the “% of total peptide bond concentration in the peak”. When more than one venom

25

protein is present in a reversed-phase fraction, their proportions can be estimated by densitometry of

26

Coomassie-stained SDS-polyacrylamide gels. On the other hand, the relative abundances of different

27

proteins contained in the same SDS-PAGE band can be estimated based on the relative ion

28

intensities of the three more abundant peptide ions associated with each protein by MS/MS analysis.

29

This elaborated approach provides a proxy of the % by weight (g/100 g) of the venom components.

30

Absolute quantitation would require accurate knowledge of ε for each venom protein. However, the

31

theoretical calculation of ε not only depends on the number of peptide bonds in the protein, but also

32

on the identity of the amino acids45. Thus, depending on the amino acid sequence, protein


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1

abundances derived from UV-Vis spectroscopic measurements may depart in an indeterminate

2

degree from their correct values making quantifications unreliable45.

3

Having demonstrated the suitability of LC-(34S)ICP-QQQ MS for the analysis and certification of

4

standards, we then sought to apply the same platform for assessing absolute quantification of the

5

venom protein components present and the corresponding protein mass purity in a real complex

6

sample such as Naja mossambica venom. Prior to those analyses, chromatographic recovery was

7

calculated, by comparing the sulfur mass of sample eluting from a chromatographic column respect

8

to the sulfur mass directly recorded by FI. Chromatographic recovery obtained for N. mossambica

9

protein content was 99 ± 1% (n=3). The total sulfur mass content corresponded to the sum of the

10

sulfur contained in the different sulfur-detected venom protein peaks (Figure 2A). This excellent

11

recovery, obtained for a real complex sample, strengthens the previous conclusion that quantitative

12

protein recoveries with the used chromatographic column are species (individual protein)

13

independent.

14

An inherent drawback of ICP-MS is that information is just “elemental” and so it is unable to

15

distinguish and identify different molecules of a mixture. Therefore, identification of the toxins

16

eluting along the chromatographic separation of a N. mossambica venom sample was carried out by

17

ESI-MS mass profiling in parallel to the ICP-MS measurements (“integrated speciation”), and

18

matching the recorded isotope-averaged molecular mass to the calculated masses for mature Naja

19

spp proteins deposited in the non-redundant NCBI database (http://www.ncbi.nlm.nih.gov) and to N.

20

mossambica venom proteins previously identified by peptide-centric venomic analysis28. This

21

workflow is displayed in Figure S4. The resolution (50000) and mass accuracy (0.2 ppm) of the

22

QToF instrument employed allowed accurate protein identification (as an example, Figure S5

23

illustrates the MS spectra for two so-identified proteins).

24

Figure 2 shows the ICP-MS and ESI-MS chromatograms obtained for the Naja mossambica venom

25

sample. Detection of up to 27 species can be observed in the inset of Figure 2A. The excellent

26

overlapping of the ICP-MS and ESI-MS profiles in the inset of Figure 2B, warranted a good peak

27

correlation between both types of chromatogram. Protein mass purity in the sample proved to be 91

28

± 1% (n=3).

29

ESI-MS protein matching allowed the straightforward transformation of the absolute S mass

30

quantified in each peak into absolute protein mass for each identified protein (see Table 2).

31

Quantitation of not-baseline-resolved peaks (e.g., Figure 2, peaks 9-10 and 14-20) was estimated by

32

vertical delimitation, and may thus be subjected to higher errors. On the other hand, for those N.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

mossambica toxins whose full-length amino acid sequence is available, the sulfur (cysteine +

2

methionine residues)/protein stoichiometry could be also derived, allowing to express the absolute

3

quantitative data as moles of toxin per gram of venom. This way of expressing the data has more

4

biological sense that g toxin/g of total venom proteins, since the number of inoculated molecules,

5

rather than their mass, is responsible for the serious pathological effects of the venom.

6

CONCLUSIONS AND PERSPECTIVES

7

In this work, we propose a straightforward methodology, based on the measurement of sulfur by

8

ICP-QQQ after HPLC separation, for the quantification in absolute terms of intact proteins by IDA

9

without the need for protein-specific standards. We have first demonstrated the suitability of the

10

proposed methodology for the absolute quantification and mass purity certification of isolated and

11

mixed protein standards. Moreover, absolute quantification of relatively complex mixtures of

12

proteins, including snake venoms, is at hand.

13

Analytical validation of the proposed ICP-MS-based approach included verification of the

14

quantitative chromatographic column recoveries for proteins of different mass and nature by using a

15

proper core-shell column. Of course, intrinsic limitations of the proposed methodology are the strict

16

requirement for good chromatographic peak resolution, and knowledge on the full-length amino acid

17

sequence of the proteins present in the sample. On the other hand, parallel mass profiling along the

18

chromatographic separation of the protein peaks by µHPLC-ESI-MS enables identification of

19

proteins by ESI-MS by resorting to existing protein databases. In the particular case of venomics,

20

this latter premise is easier by the increasing tendency of combining locus-resolved venom

21

proteomic46,47 and venom gland transcriptomic48 analyses in integrative venomic workflows. The

22

addition of simultaneous µHPLC-ICP-MS helps substantially to simplify the complexity of real life

23

samples (e.g. venoms).

24

In conclusion, a broad range of applications can be foreseen for the synergic MS methodology

25

proposed here for the absolute quantification of proteins; first, it may be invaluable to certify and

26

quantify isotopically labeled peptides and proteins (extensively needed and used as internal standards

27

in molecular MS-based quantitative proteomics49,50); second, the combined elemental and molecular

28

MS-based chromatographic detection could also be very useful to simplify the needed quantitative

29

studies of relatively complex mixtures of proteins. In this sense, in the field of venomics, integrating

30

the absolute quantitation of the toxins of a venom proteome with the ever increasing analytical

31

capacity of the different omic technologies, could have a dramatic impact in advances in venomics51,


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1

toxicovenomics52, or ecological and evolutionary venomics53. Of course, the elemental/molecular

2

MS approach facilitates simpler visions (via ICP-MS) of molecular toxicology problems.

3 4

ACKNOWLEDGMENTS

5

Authors wish to thank Agilent and Agilent Foundation for the generous technical and financial

6

support, and Quality Assistance for financial support. F.C.C. thanks the Ministry of Economy and

7

Competitiveness of Spain (grant BES-2014-068032). S.D.F. thanks FICYT (Principado de Asturias)

8

for the ‘‘Severo Ochoa’’ grant (BP13094). Also, the assistance during ESI-QToF analyses of Dr.

9

Sergio Cueto Diaz, from Scientific Services of University of Oviedo, is gratefully acknowledge.

10

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TABLES

1 2 3

Table 1. Chromatographic recoveries of the assayed BOC-Met and protein standards. Uncertainty

4

corresponds to 1 standard deviation (n=2).

5

Compound

Recovery (%)

BOC-Met

96.2 ± 3.3

cytochrome C

99.4 ± 3.2

β-casein

99.1 ± 2.7

BSA

98.1 ± 2.7

transferrin

98.1 ± 3.4

Intact mAb

99.6 ± 3.5

6



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Page 18 of 21

1

Table 2. Comparison of protein mass purity for cytochrome C, transferrin and BSA with the theoretical ones

2

provided by manufacturer. Uncertainty corresponds to 1 standard deviation (n=3).

3

Compound

Protein mass purity (manufacturer)

Experimental Protein mass purity purity

cytochrome C

≥ 95 %

94 ± 2

BSA

≥ 98 %

96 ± 2

transferrin

≥ 95 %

94 ± 2

4 5


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1

Table 3. Matching of the masses of protein peaks from Naja mossambica venom to known protein families.

2

Closest available protein species, estimated exact mass, and calculated concentration, are listed. Uncertainty

3

corresponds to 1 standard deviation (n=3).

4

Peak

Family

Closest homolog

MW (Da)

µmol protein/g venom sample

1

3FTx

-

7064.2

1.99 ± 0.06

2

3FTx

∼P29179

7417.4

0.471 ± 0.066

3

3FTx

∼P29179

7451.6

0.325 ± 0.040

4

3FTx

∼P01420

6892.4

1.10 ± 0.13

5

3FTx

∼Q9W6W6

7786.4

< 0.1

6

3FTx

∼P01452

7277.3

0.680 ± 0.050

7

3FTx

∼P01452

7306.3

0.668 ± 0.057

8

3FTx

-

7246.2

1.35 ± 0.10

9

3FTx

P25517

6832.4

5.09 ± 0.28

10

3FTx

P01452

6704.3

19.0 ± 0.8

11

3FTx

-

6686.3

0.183 ± 0.035

12

3FTx

-

6829.3

0.220 ± 0.039

13

3FTx

-

6687.3

< 0.1

14

PLA2

P00604

13280.9

7.76 ± 0.32

15

3FTx

P01470

6882.4

9.54 ± 0.27

16

3FTx

P25517

6813.3

16.2 ± 0.4

17

3FTx

P01467

6814.3

27.8 ± 0.8

18

3FTx

∼P01469

7046.4

5.13 ± 0.26

19

PLA2

P00604

13237.8

3.40 ± 0.14

20

PLA2

P00002

13196.6

7.35 ± 0.33

21

PLA2

-

13179.7

0.805 ± 0.049

22

Minor

-

42000

0.102 ± 0.009

23

Endonuclease

-

30000

0.619 ± 0.050

24

SVMP

Q10749

46700

0.165 ± 0.009

25

SVMP

Q10750

46700

0.264 ± 0.013

26

SVMP

Q10751

46700

0.097 ± 0.006

27

SVMP

Q10752

46700

0.257 ± 0.006

5 6



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

FIGURES

2 3

Figure 1. (A) Mass flow chromatogram of Met-BOC and BSA protein mixture (in order of elution) (B)

4

Mass flow chromatogram of Met-BOC and Intact mAb mixture (in order of elution) (C) Mass flow

5

chromatogram of Met-BOC, cytochrome C, transferrin and BSA (in order of elution). Arrows indicate a

6

perturbation in the first minutes of the chromatogram corresponding to signal instability due to the

7

injection and/or sulfur-containing impurities.

8

9 10


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1

Figure 2. (A) ICP-MS mass flow chromatogram of Naja mossambica venom. All the venom protein species

2

eluted between 20 and 50 min. The 27 chromatographic peaks observed are numbered in the inset of the

3

figure. (B) ESI-MS chromatogram of Naja mossambica venom sample. Inset, peak correlation of ICP-QQQ

4

(front) and ESI-QToF (back) spectra in the range of 22-39 min, corresponding to the elution time of the 3FTx

5

and PLA2 protein species. The observed excellent peak patterns matching enabled correlating molecular peak

6

identity and elemental S quantitation.

7

8

9


Elemental Mass Spectrometry for Absolute Intact Protein Quantification

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