**7. Metallomics studies involving HPLC coupled to ICP-MS**

In the last years, many soybean varieties have been genetically modified for adaptation to different geographical regions, to increase quality and productivity. Due to these genetic modifications, the proteins composition and profile can be affected, causing changes in the species proteome [91-92]. As previously described, the knowledge of the soybean genotype alone does not show enough information about the protein modifications due to environ‐ mental interactions. For better understanding of the consequences of a genetic manipulation, the elucidation of the protein map composition is necessary because it is directly related to the phenotype [93]. Since the proteome can be affected, it is assumed that the metallome can also be affected somehow by the genetic modification [94].

The metallome is defined as the entirety of metals and metalloid species, present in a cell or tissue type [95]. Deciphering the metallome provides information such as: (i) how an ele‐ ment is distributed among the cellular compartments; (ii) its coordination environment, in which the biomolecule is incorporated or by which bioligand it is complexed, and (iii) the

concentration of the individual metal species present [96]. The majority of metals present in biological fluids and organs are linked to proteins, called metalloproteins. It is believed that every third protein require a metal cofactor, such as Cu, Fe and Zn, to develop their func‐ tions correctly [97]. The determination of an organism metallome involves separation techni‐ ques associated to microanalytic processes, such as mass spectrometry. These are the two key steps for general proteomics: separation and posterior identification of the proteins [80].

Metallomics studies were already performed in our group, being that one involving compa‐ rative metallomics of transgenic and non-transgenic soybean seeds [94], the first published in the literature. Soybean proteins were separated using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), tryptically digested, characterized using matrix assisted la‐ ser desorption ionization - quadrupole time of flight – mass spectrometry (MALDI-Q-TOF-MS) and mapped using synchrotron X-ray fluorescence radiation (SR-XRF). The following metallic ions were found: Ca(II), Cu(II), Fe(II), Mn(II), Ni(II) and Zn(II), and the quantitative profile was acquired using atomic absorption spectrometry, showing changes in metal con‐ tents of transgenic and non-transgenic soybean seeds. Although promising results could be found in this study, the canonical analytical approaches for proteomics (such as 2-D PAGE) and metabolomics studies usually do not consider the existence of metal complexes with proteins and metabolites.

In this way, the use of high performance liquid chromatography (HPLC), an analytical technique used to separate a mixture in solution in its individual components, should be considered. Distinctly compared from 2-D PAGE, HPLC is based on different proteinsurface interactions [98]. The principal HPLC separation mechanisms used for bioinor‐ ganic studies include size exclusion chromatography (SEC), reversed-phase (RP) and ionexchange (IEX) chromatography, and because of the complex nature of the metalmolecule interaction, a combination of these mechanisms is often necessary to identify the elemental species correctly.

Together with HPLC, an element-specific detector must be used, and since the 80's, induc‐ tively coupled plasma mass spectrometry (ICP-MS) is being widely applied for studying ele‐ ments at low concentrations. It is robust for multielementar determinations, allowing to reach extremely low detection limits and giving isotopic information for identification and quantification of the species, besides being easily coupled to classic separation techniques, such as HPLC, readily realized since the chromatographic flow (0.5-1.0 mL min-1) is compat‐ ible to common ICP-MS nebulizers [97,99].

The excitation source of this technique is argon inductively coupled plasma, which is used to form ions which are transferred to a high vacuum region through an interface containing small orifices. Ions are focalized using ionic lenses and directed to the mass spectrometer in order to be separated by *m/z* ratio. The *m/z* ratio analyzer generally used in ICP-MS is a quadrupole, ideal for quantitative analysis [100].

The elements detected using ICP-MS include metal coordination complexes with larger pro‐ teins and metallothioneins, as well as selenoproteins and metal/semi-metals linked to carbo‐ hydrates. A great amount of examples are found in the literature where ICP-MS is used to detect and quantify metallic ions bounded to biomolecules.

concentration of the individual metal species present [96]. The majority of metals present in biological fluids and organs are linked to proteins, called metalloproteins. It is believed that every third protein require a metal cofactor, such as Cu, Fe and Zn, to develop their func‐ tions correctly [97]. The determination of an organism metallome involves separation techni‐ ques associated to microanalytic processes, such as mass spectrometry. These are the two key steps for general proteomics: separation and posterior identification of the proteins [80].

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Metallomics studies were already performed in our group, being that one involving compa‐ rative metallomics of transgenic and non-transgenic soybean seeds [94], the first published in the literature. Soybean proteins were separated using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), tryptically digested, characterized using matrix assisted la‐ ser desorption ionization - quadrupole time of flight – mass spectrometry (MALDI-Q-TOF-MS) and mapped using synchrotron X-ray fluorescence radiation (SR-XRF). The following metallic ions were found: Ca(II), Cu(II), Fe(II), Mn(II), Ni(II) and Zn(II), and the quantitative profile was acquired using atomic absorption spectrometry, showing changes in metal con‐ tents of transgenic and non-transgenic soybean seeds. Although promising results could be found in this study, the canonical analytical approaches for proteomics (such as 2-D PAGE) and metabolomics studies usually do not consider the existence of metal complexes with

In this way, the use of high performance liquid chromatography (HPLC), an analytical technique used to separate a mixture in solution in its individual components, should be considered. Distinctly compared from 2-D PAGE, HPLC is based on different proteinsurface interactions [98]. The principal HPLC separation mechanisms used for bioinor‐ ganic studies include size exclusion chromatography (SEC), reversed-phase (RP) and ionexchange (IEX) chromatography, and because of the complex nature of the metalmolecule interaction, a combination of these mechanisms is often necessary to identify

Together with HPLC, an element-specific detector must be used, and since the 80's, induc‐ tively coupled plasma mass spectrometry (ICP-MS) is being widely applied for studying ele‐ ments at low concentrations. It is robust for multielementar determinations, allowing to reach extremely low detection limits and giving isotopic information for identification and quantification of the species, besides being easily coupled to classic separation techniques, such as HPLC, readily realized since the chromatographic flow (0.5-1.0 mL min-1) is compat‐

The excitation source of this technique is argon inductively coupled plasma, which is used to form ions which are transferred to a high vacuum region through an interface containing small orifices. Ions are focalized using ionic lenses and directed to the mass spectrometer in order to be separated by *m/z* ratio. The *m/z* ratio analyzer generally used in ICP-MS is a

The elements detected using ICP-MS include metal coordination complexes with larger pro‐ teins and metallothioneins, as well as selenoproteins and metal/semi-metals linked to carbo‐

proteins and metabolites.

Relationships

596

the elemental species correctly.

ible to common ICP-MS nebulizers [97,99].

quadrupole, ideal for quantitative analysis [100].

The attempts to avoid metal-ligand denaturation make the SEC mechanism the most used for metallomics studies [97]. It separates molecules according to their hydrodynamic vol‐ ume, determined by their Stokes ratio. It results in the partial exclusion of analytes that pass through defined size pores due to their molecular sizes [101]. When the mobile phase passes through the column, those particles with small hydrodynamic volumes are transported through a larger path because they equilibrate in the pores more frequently than the ones with higher hydrodynamic volumes, resulting in separation. Elution volume is determined by the molecule size, directly related to their molecular weight, so this volume can be used to determine the molecular weight of an unknown compound. For carrying out that task, the relation between molecular weight and elution volume, obtained empirically by inject‐ ing standards with known molecular weight and measuring their elution volumes, must be known [102-103].

SEC is especially suitable for separation of element species presenting limited stability fre‐ quently found in protein-rich matrices. The main advantages of SEC are simplicity of appli‐ cation, tolerance to biological matrices, compatibility of mobile phases with specific demands of certain biological samples and the possibility of estimation of molecular weights of the compounds. It is widely used for protein separation, including soybean proteins and, although considered a low resolution method [104], it is often applied as the first separation method of fractions containing metallo-biomolecules of interest followed by another separa‐ tion step with element-specific detection or MS identification. Its uses alone is very helpful to study the distribution of elements in different molecular weight fractions, and the cou‐ pling SEC-ICP-MS is being accepted as a hyphenated technique for speciation studies to evaluate the association of elements to compounds present in the sample. These studies are considered the initial point for a deeper evaluation of the nature of the species found.

In a work developed in our group [105], a comparison between elution profiles from trans‐ genic and non-transgenic soybean seeds was carried out, using SEC coupled to high resolu‐ tion ICP-MS. The elution profiles were similar between the samples, and the conclusions are in agreement to the ones discussed by reference [106]. It was found that areas of the most abundant peaks for Cu and Fe in transgenic soybean seeds were 3- and 2-fold higher, re‐ spectively, than those found in non-transgenic samples. This, summed up with total element analysis results in the same article, where the concentrations of Cu and Fe had statistically significant differences between transgenic and non-transgenic soybean seeds, could lead to the conclusion that Cu and Fe are associated with compounds more expressed in transgenic soybean seeds.

Reference [107] used different parts of the soybean plants to analyze Se elution profile using SEC-ICP-MS. The authors concluded that the bean had the most interesting profile, since it absorbed most of the Se from the shoots and presented a very intense peak for this element at higher molecular weight fraction. These data showed that the soybean plants convert se‐ lenite (used to enrich the plant) to high molecular weight species, which, according to the authors, can add nutritional value to the plant. Another work from the same group [108]

used reversed phase coupled to ICP-MS to study the Se-Hg antagonism, and they found that in plants enriched with Hg, more Se was assimilated, indicating a possible protective re‐ sponse mechanism to the Hg.

As already commented, it is known that purity of peaks in SEC is poor, and even if a single species of a given element is present, matrix components may co-elute. They are invisible to the element specific detector, but if the goal is the identification of the organic specie linked to the element, they will be detected by the organic MS instrument. Also, matching the elu‐ tion volume with a standard in this case is not definitive, due to the small number of theo‐ retical plates found in SEC. For these reasons, SEC is usually followed by a second chromatographic separation (2nd dimension) using an orthogonal separation mechanism, such as ion-exchange, reversed-phase or hydrophilic interaction chromatography, before the identification of the components.

Multidimensional liquid chromatography is an efficient tool and an alternative procedure for the classic methods based on unidimensional HPLC. The multidimensional chromatog‐ raphy can be carried out *online* or *off-line*. In the *off-line* mode, fractions eluted from the 1st dimension are collected manually or using a fraction collector, and then are re-injected in the second chromatographic column. *Online* techniques are automated using a selector valve, which can enhance reliability and sample processing. The limitation here is that the mobile phases used for both dimensions must be compatible [109].

The selectivity in a multidimensional system can be enhanced only if the chromatographic dimensions are based in different separation mechanisms. The second dimension must not decrease the resolution obtained using the previous one. For the separation mechanisms to be different, the columns must have different stationary phases, allowing the less efficient separation attained in the first dimension to be improved in the second [110].

In the case of multidimensional liquid chromatography [103,111] coupled to ICP-MS, frac‐ tions isolated mainly using SEC, can be fractionated again using an independent separation mechanism to provide more detailed results, and also to attain metal species pure enough to be characterized using molecular mass spectrometry.

Many stationary phases can be used for a second chromatographic dimension. Among then, reversed phase (RP) [111], the most popular liquid chromatography separation mechanism, should be highlighted. It has great efficiency and is able to separate a great range of com‐ pounds with different polarities. The separation is obtained through partition of the analyte between a non-polar stationary phase and a polar mobile phase.

Ion exchange chromatography [112] (IEX) can also be used to separate biomolecules based in charge differences. It can be considered a highly selective technique, able to separate, for example, proteins differing in only one charged group. It is a widely used technique in bio‐ separations, since peptides, proteins, nucleic acids and related biopolymers have ionizable chemical domains, making them susceptible to enhancement or diminishment of their charges as a function of pH and ionic strength changes. It can be used to separate large bio‐ molecules, with more than 60 kDa.

Finally, polar compounds can be efficiently separated using polar/hydrophilic stationary phases using normal phase aqueous chromatography (aqNPC), also called hydrophilic interaction chro‐ matography (HILIC) [113]. Here, retention times tend to be longer as high as is the hydrophobicity of the solutes, indicating potential for small metallic complexes separation.

used reversed phase coupled to ICP-MS to study the Se-Hg antagonism, and they found that in plants enriched with Hg, more Se was assimilated, indicating a possible protective re‐

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

As already commented, it is known that purity of peaks in SEC is poor, and even if a single species of a given element is present, matrix components may co-elute. They are invisible to the element specific detector, but if the goal is the identification of the organic specie linked to the element, they will be detected by the organic MS instrument. Also, matching the elu‐ tion volume with a standard in this case is not definitive, due to the small number of theo‐ retical plates found in SEC. For these reasons, SEC is usually followed by a second chromatographic separation (2nd dimension) using an orthogonal separation mechanism, such as ion-exchange, reversed-phase or hydrophilic interaction chromatography, before the

Multidimensional liquid chromatography is an efficient tool and an alternative procedure for the classic methods based on unidimensional HPLC. The multidimensional chromatog‐ raphy can be carried out *online* or *off-line*. In the *off-line* mode, fractions eluted from the 1st dimension are collected manually or using a fraction collector, and then are re-injected in the second chromatographic column. *Online* techniques are automated using a selector valve, which can enhance reliability and sample processing. The limitation here is that the

The selectivity in a multidimensional system can be enhanced only if the chromatographic dimensions are based in different separation mechanisms. The second dimension must not decrease the resolution obtained using the previous one. For the separation mechanisms to be different, the columns must have different stationary phases, allowing the less efficient

In the case of multidimensional liquid chromatography [103,111] coupled to ICP-MS, frac‐ tions isolated mainly using SEC, can be fractionated again using an independent separation mechanism to provide more detailed results, and also to attain metal species pure enough to

Many stationary phases can be used for a second chromatographic dimension. Among then, reversed phase (RP) [111], the most popular liquid chromatography separation mechanism, should be highlighted. It has great efficiency and is able to separate a great range of com‐ pounds with different polarities. The separation is obtained through partition of the analyte

Ion exchange chromatography [112] (IEX) can also be used to separate biomolecules based in charge differences. It can be considered a highly selective technique, able to separate, for example, proteins differing in only one charged group. It is a widely used technique in bio‐ separations, since peptides, proteins, nucleic acids and related biopolymers have ionizable chemical domains, making them susceptible to enhancement or diminishment of their charges as a function of pH and ionic strength changes. It can be used to separate large bio‐

mobile phases used for both dimensions must be compatible [109].

be characterized using molecular mass spectrometry.

molecules, with more than 60 kDa.

between a non-polar stationary phase and a polar mobile phase.

separation attained in the first dimension to be improved in the second [110].

sponse mechanism to the Hg.

Relationships

598

identification of the components.

Concerning multidimensional chromatographic separations and soybeans, a recent work from reference [107] used IEX as second dimension for the separation of proteins from sele‐ nium-enriched soybean. Here, the target was only Se, and the 26 fractions collected from the second dimension were pure enough to allow the identification of a considerable number of proteins in the soybean databank.

In our group [114], IEX was also used as second dimension, generating a number of differ‐ ent fractions for both transgenic and non-transgenic soybean seeds used in that research. Taking as an example cobalt, the SEC separation (Figure 2a) provided 3 peaks, divided into F1, F2, and F3. When F3 is separated again using IEX (Figure 2b), the wide peak found using SEC was separated into two narrow peaks, showing that the separation resolution in‐ creased.

**Figure 2.** Chromatograms for UV absorption at 280 nm (\_\_\_) and Co signal in the ICP-MS (\_\_\_) for (a) SEC, separated in three fractions: F1, F2 and F3; and (b) IEX separation of F3 using transgenic soybean seeds [modified from supplemen‐ tary material of reference 114].

Nowadays, mass spectrometry associated to bioinformatics has become essential in studies involving proteins, not only due to their sensitivity, but also to the total of information that can be obtained [69]. Electrospray ionization (ESI) is the most used technique for protein identification, allowing the formation of ions in the gas phase using a soft ionization proc‐ ess, making possible the analysis of non-volatile and thermolabile compounds [115]. As a consequence, ESI facilitated the analysis of large biomolecules, as well as drugs and their metabolites.

To improve metallomics information concerning transgenic and non-transgenic soybean seeds, our group [114] used the multidimensional chromatography strategy, as pointed out previously in this text. Total amounts of Fe and Cu were already found to be higher in transgenic soybeans, and in an attempt to link these metals to proteins, an ESI-MS/MS analysis was carried out. As results, more

than 20 proteins were identified, encompassing 4 different functional categories. Among them, βconglycinin, a protein previously associated to metals, was identified in three fractions, and one metalloprotein that binds Fe, lypoxigenase 1, was found in a high molecular weight fraction, the on‐ ly fraction where an Fe peak was separated.
