**5. Two-dimensional gel electrophoresis: 2-D PAGE**

In 2-D PAGE technique, the proteins are first separated on the basis of their net charges by isoelectric focusing (IEF) and then separated on the basis of their molecular mass by polya‐ crylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) [67].

The combination of 2-D PAGE and mass spectrometry is one excellent strategy to obtain proteomic maps [78]. Furthermore, taking into account that we were finding some differen‐ tial proteins when investigating transgenic and non-transgenic soybean seeds, this techni‐ que showed itself to be excellent for this task. In this sense, our experience in terms of this kind of study will be commented.

Reference [79] used the combination of 2-D PAGE and mass spectrometry for obtaining a proteomic map for transgenic soybean seeds. The literature reports [12] that the number of protein spots present in the linear pH range from 4 to 7 was higher than the number of spots present in the linear range from 3 to 10 for transgenic soybean seeds. Therefore, for the range from 4 to 7, a higher number of spots were selected when compared with the 3-10 range. As result, a total of 192 proteins from transgenic soybean seeds were identified, 179 of them identified within the pH range from 4 to 7, and 13 of them identified within the 3-10 pH range. Regarding the pH range from 4 to 7, 49% of the spots present in the gel were identified in the database showing good efficiency with a similar study involving soybean published in the literature [80]. Regarding their biological functions, 50% were related to storage function, 18% related in growth/cell division process, 9% involved in metabolic/ energy process, 6% related to protein transport, 4% corresponding to proteins involved in the disease/defense category and 21% in the category of non-classified proteins.

The application of the 2-D PAGE technique in comparative proteomic studies can lead to some problems due to the intrinsic characteristics of the electrophoretic systems such as sample preparation strategies, the natural variations when considering biological systems, gel-to-gel variance, labor intensiveness and possible identification of several proteins from one spot [67,81]. In this sense, 2-DE technologies need to be evaluated critically.

In a pioneering work, reference [66] evaluates some parameters that influence the compari‐ sons of the protein map after different gel runs, establishing comparative image analysis af‐ ter 2-D PAGE of transgenic and non-transgenic soybean seeds for identifying possible differences in protein expression. In that work, two pH ranges were used: 3-10 and 4-7. For improving accuracy, image treatments were made by the same analyst and concomitantly carried out for each pair of gels in the same electrophoretic run (4 pairs of gels with the opti‐ mized loaded mass) for avoiding possible variations between evaluations of the gel images.

As a result, the enzyme cp4 EPSPS was identified using the SDS-PAGE technique and using either trypsin or chymotrypsin as a cleavage enzyme. However, trypsin showed the best re‐ sults in terms of score and coverage (as a percentage). Moreover, the enzyme was identified in the database containing sequences from the soil bacterium *Agrobacterium* sp, the origin of the gene used in genetic modification. This method proved to be simple and very efficient,

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

In 2-D PAGE technique, the proteins are first separated on the basis of their net charges by isoelectric focusing (IEF) and then separated on the basis of their molecular mass by polya‐

The combination of 2-D PAGE and mass spectrometry is one excellent strategy to obtain proteomic maps [78]. Furthermore, taking into account that we were finding some differen‐ tial proteins when investigating transgenic and non-transgenic soybean seeds, this techni‐ que showed itself to be excellent for this task. In this sense, our experience in terms of this

Reference [79] used the combination of 2-D PAGE and mass spectrometry for obtaining a proteomic map for transgenic soybean seeds. The literature reports [12] that the number of protein spots present in the linear pH range from 4 to 7 was higher than the number of spots present in the linear range from 3 to 10 for transgenic soybean seeds. Therefore, for the range from 4 to 7, a higher number of spots were selected when compared with the 3-10 range. As result, a total of 192 proteins from transgenic soybean seeds were identified, 179 of them identified within the pH range from 4 to 7, and 13 of them identified within the 3-10 pH range. Regarding the pH range from 4 to 7, 49% of the spots present in the gel were identified in the database showing good efficiency with a similar study involving soybean published in the literature [80]. Regarding their biological functions, 50% were related to storage function, 18% related in growth/cell division process, 9% involved in metabolic/ energy process, 6% related to protein transport, 4% corresponding to proteins involved in

crylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) [67].

the disease/defense category and 21% in the category of non-classified proteins.

one spot [67,81]. In this sense, 2-DE technologies need to be evaluated critically.

The application of the 2-D PAGE technique in comparative proteomic studies can lead to some problems due to the intrinsic characteristics of the electrophoretic systems such as sample preparation strategies, the natural variations when considering biological systems, gel-to-gel variance, labor intensiveness and possible identification of several proteins from

In a pioneering work, reference [66] evaluates some parameters that influence the compari‐ sons of the protein map after different gel runs, establishing comparative image analysis af‐ ter 2-D PAGE of transgenic and non-transgenic soybean seeds for identifying possible differences in protein expression. In that work, two pH ranges were used: 3-10 and 4-7. For improving accuracy, image treatments were made by the same analyst and concomitantly

without needing sample prefractionation using chromatographic columns [77].

**5. Two-dimensional gel electrophoresis: 2-D PAGE**

kind of study will be commented.

Relationships

592

In relation to detection and selection of the protein spots, the choice of the parameters of im‐ age analysis is extremely important. Differences between manual and automatic detection of the spots were obtained, showing the importance in editing the images to avoid erroneous interpretations not only in terms of the quantities of the detected spots, but also in terms of the intensities and/or volume of each protein detected.

The matching study is of utmost importance for those ones where the target is to find possi‐ ble changes in protein expression as well as to establish the similarities in protein distribu‐ tion in sets of gels. For gels obtained in the same run and within 3 to 10 and 4 to 7 pH ranges, 163±37 (79±4% match) and 287±28 spots (77±2% match) were respectively obtained from 4 pairs of gels (transgenic *x* non-transgenic). However, when gels were obtained from different runs, even considering the same sample (transgenic seeds), high variation was de‐ tected in terms of matches (39±6% and 58±13% for 3–10 and 4–7 pH ranges gels, respective‐ ly). Similar results for non-transgenic seeds were obtained (40±10% and 62±18% for 3–10 and 4–7 pH gel ranges, respectively). In this way, it is preferable to acquire the gels in the same run in order to produce high matches. The use of these procedures points out that elimina‐ tion of gel-to-gel variation is mandatory in image analysis.

Proteins were considered as up or down regulated when the ratio between spot volume and/or intensity for non-transgenic and transgenic soybean seeds changed from < 0.55 to >1.8 (*ca*. 90% variation). Thus, 3 and 7 spots from 3 to 10 and 4 to 7 pH ranges were respec‐ tively highlighted and characterized by MALDI-QTOF MS. From this total, 8 proteins were identified as: glycinin G2/A2B1 precursor, glycinin G1 precursor, α-subunit of β-conglycinin (03 spots), allergen Gly mBd 28 K (fragment), actin (fragment) and sucrose binding protein.

Then, it is easy to observe that well optimized conditions for acquiring images from 2D gels are an important tool in the identification of possible biomarkers for genetically modified organisms.
