**4.1 Plasma sample preparations**

Sample preparation is an important step that influences the separation of proteins with 2- DE. A common problem in most biological samples is the presence of salt ions. In 2-DE, salt concentrations >10 mM affects the isoelectric focusing step and markedly reduces the effectiveness of the charge separation. There are several easy ways to remove salts, e.g. by precipitation of the proteins or by gelfiltration in small desalting columns. Plasma samples are possible to analyze directly with 2-DE since the high protein concentration allows a simple dilution of the sample to lower the salt concentration. However, as illustrated in figure 7, a desalting step besides the dilution still improves the protein pattern. Another well-known problem with biological fluids is the presence of a few highly abundant proteins that may prevail over the low abundant proteins. In plasma, albumin and immunoglobulin G (IgG) constitutes a very large proportion (about 75%) of the total protein loaded on the gels. This may lead to the proportion of low abundant proteins being below the detection limit. Also, the staining of the abundant proteins may interfere with proteins with similar molecular mass and pI. It is therefore advisable to remove albumin and IgG and there are several commercial removal kits available, usually based on antibodies directed towards albumin and IgG. As shown in figure 7, such sample preparation step removes a large fraction of these proteins and increases the proportion of the other proteins in the sample. However, it is important to realize that this step also introduces unspecific removal of proteins and it is our experience that this unwanted loss of proteins varies considerable between the different available removal kits.

Untreated plasma (left), desalted plasma (middle) and desalted plasma after removal of albumin and immunoglobulin G (IgG), (right), were separated with 2-DE and silver stained. Arrows indicate protein or protein clusters with increased abundance after treatment compared to untreated plasma. The rectangle indicate an area with improved resolution after desalting. Positions for albumin and IgG chains are indicated with rings.

Plasma contains a wide variety of proteins, many of which are not detectable with 2-DE without further fractionation. In view of the lipid metabolism, important sub-fractions of plasma to study are the lipoproteins. As shown in table 1, one protein that interacts with

Sample preparation is an important step that influences the separation of proteins with 2- DE. A common problem in most biological samples is the presence of salt ions. In 2-DE, salt concentrations >10 mM affects the isoelectric focusing step and markedly reduces the effectiveness of the charge separation. There are several easy ways to remove salts, e.g. by precipitation of the proteins or by gelfiltration in small desalting columns. Plasma samples are possible to analyze directly with 2-DE since the high protein concentration allows a simple dilution of the sample to lower the salt concentration. However, as illustrated in figure 7, a desalting step besides the dilution still improves the protein pattern. Another well-known problem with biological fluids is the presence of a few highly abundant proteins that may prevail over the low abundant proteins. In plasma, albumin and immunoglobulin G (IgG) constitutes a very large proportion (about 75%) of the total protein loaded on the gels. This may lead to the proportion of low abundant proteins being below the detection limit. Also, the staining of the abundant proteins may interfere with proteins with similar molecular mass and pI. It is therefore advisable to remove albumin and IgG and there are several commercial removal kits available, usually based on antibodies directed towards albumin and IgG. As shown in figure 7, such sample preparation step removes a large fraction of these proteins and increases the proportion of the other proteins in the sample. However, it is important to realize that this step also introduces unspecific removal of proteins and it is our experience that this unwanted loss of proteins varies

Untreated plasma (left), desalted plasma (middle) and desalted plasma after removal of albumin and immunoglobulin G (IgG), (right), were separated with 2-DE and silver stained. Arrows indicate protein or protein clusters with increased abundance after treatment compared to untreated plasma. The rectangle indicate an area with improved resolution

Plasma contains a wide variety of proteins, many of which are not detectable with 2-DE without further fractionation. In view of the lipid metabolism, important sub-fractions of plasma to study are the lipoproteins. As shown in table 1, one protein that interacts with

after desalting. Positions for albumin and IgG chains are indicated with rings.

**4. Methodological considerations and improvements of 2-DE and** 

considerable between the different available removal kits.

Fig. 7. Preparation of plasma for 2-DE

**MALDI-TOF MS** 

**4.1 Plasma sample preparations** 

nanoparticles is apo A-I, the major constituent of HDL. This implicates the need of more investigations of HDL as a possible target of nanoparticles that may influence the cholesterol metabolism and increase the risk of cardiovascular disease. HDL can be isolated based on density, size or protein content (e.g. apo A-I) using ultracentrifugation, size-exclusion chromatography or immune-affinity chromatography, respectively, each technique with its own merits and drawbacks. Thus, the rather harsh conditions during ultracentrifugation in high salt gradients may remove weakly associated proteins while the rather mild conditions during chromatography may favor unspecific co-purification of proteins with the cholesterol particle. We have previously mapped the protein content of HDL isolated by two-step density gradient ultracentrifugation (Karlsson et al., 2005). In this study we have compared ultracentrifugation and anti-apo A-I affinity chromatography to isolate HDL from the same plasma sample. As shown in figure 8, more proteins were obviously identified in immune-affinity purified HDL. However, some of the proteins must be considered as possible plasma contaminants as they were not, as e.g. the apolipoproteins, enriched in the HDL fraction.

Fig. 8. Subfractionation of plasma with regard to HDL Plasma (left), HDL purified according to apo A-I with immunoaffinity chromatography (middle) and HDL isolated according to density with ultracentrifugation (right) were separated with 2-DE and silver stained. Proteins were identified with mass spectrometry.

#### **4.2 Protein detection**

Gel-separated proteins can be visualized by several commonly used staining methods, including dyes (e.g. Coomassie Brilliant Blue and colloidal Coomassie), metals (e.g. silver staining) and fluorescent probes (e.g. Sypro staining and Cy-dyes) (Rabilloud, 2000). The stains interact differently with the proteins and have different limitations with regard to sensitivity, linear range, compatibility with mass spectrometry and type of proteins that stain best. In general, for staining of complex protein samples, silver staining can be considered the most sensitive technique (1-5 ng protein) and Coomassie Brilliant blue the least (50-100 ng) while the sensitivity of colloidal coomassie and the fluorescent dyes are in between. However, it is important to bear in mind that the different stains interact differently with the proteins and therefore one protein may stain very well with one staining method but not with the other (Fig. 9). For example, silver ions react with negatively charged groups and therefore stain glycoproteins containing negative sialic acid very well.

Two-Dimensional Gel Electrophoresis and Mass

**4.3.1 Alternative digestion of samples** 

Spectrometry in Studies of Nanoparticle-Protein Interactions 347

positive results. There are several approaches to consider for improving the data from the

One of the most widely used ways to digest proteins before MS analyses is by trypsin, which cleaves C-terminally of lysine and arginine (not followed by proline). Although lysine and arginine often are distributed in the protein sequences in a way that provides sufficient number of peptides for identification after trypsin cleavage, this is not always so. Furthermore, it is sometimes necessary to use alternative digestion protocols in order to find specific peptides to e.g. characterize differences between protein isoforms. In these cases alternative enzymes, e.g. Asp-N (cleaves N-terminally of aspartic acid and cystein), Glu-C (C-terminally of glutamic acid) or chemical induced cleavage by CNBr that hydrolyzes Cterminally of methionine, is needed. In this study we used Asp-N as a complement to trypsin when identifying the plasma protein serum amyloid A4 (SAA4). This combined digestion approach generated almost 95 % sequence coverage of the protein (figure 10). SAA4 is a constitutively expressed protein which can be found in HDL as differently charged isoforms (fig 14, Karlsson et al., 2005). One explanation to these isoforms could be a small truncation of SAA4 in which one lysine and one tyrosine is removed C-terminally and thereby making the protein more acidic (Farwig et al., 2005). However, by using Asp-N we were able to detect both the intact C-terminal and the intact N-terminal peptide that were not possible after trypsin digestion, ruling out the presence of truncated SAA4 in our sample (figure 10). Besides the use of Asp-N to study SAA4, we have also used Glu-C to analyze apolipoprotein A-I (figure 6) and CNBr to study serum amyloid A-1/2 isoforms (figure 13).

Fig. 10. Alternative digestion to improve sequence coverage of Serum amyloid A4 (SAA4).

Sequence coverage obtained through peptide mass fingerprinting with MALDI-TOF mass spectrometry by the use of trypsin and endoproteinase Asp-N as indicated by the lines. Sequence coverage (without the signal peptide in position 1-18) was 79.5 % with trypsin and

Peptide samples after in-gel digestion can be cleaned by adsorption to C-18 containing pipette tips (ZipTip®). This clean-up procedure done manually is rather time-consuming but is absolutely necessary before electrospray-quadrupole MS. On the other hand, with a MALDI-TOF instrument, being more insensitive to salts and other contaminants, it is not that obvious. To investigate the possible advantage with ziptip cleaning before MALDI-TOF MS we picked 17 sypro stained proteins after 2-DE. The proteins were digested with trypsin and an aliquot of the obtained peptide solution was purified by ZipTip® (50% ACN elution solution according to the protocol recommended by the supplier), mixed with the matrix CHCA and spotted on the MALDI-target plate and another aliquot of the peptide solution was mixed directly with the matrix and spotted on the same plate. All samples were then

57.1 % with Asp-N. The combined sequence coverage was 93.8%.

**4.3.2 Peptide sample cleaning** 

analyses, such as digestion protocol, purification of peptides and choice of matrix.

One the contrary, Sypro ruby that binds to proteins through hydrophobic interactions stains hydrophilic glycoproteins quite poorly. Being most sensitive, silver staining is obviously very useful for proteomic approaches. However, the advantage with silver is hampered by its rather low linear range, making it less suitable for quantification than the other staining techniques. For plasma analyses we have therefore adopted a double staining strategy. As illustrated in figure 9, the 2-DE gel is first stained with Sypro Ruby and the proteins are quantified within a high dynamic range. The gel then can be destained and restained by silver to detect additional proteins. As these additional proteins are less abundant their intensities usually are within the limited linear range of the silver staining technique. The proteins may then be selected for MS analyses. As the sample preparation protocol is more time-consuming for silver stained gels than Sypro stained gels it is convenient to pick as many proteins as possible after the first staining step. In this step it is also important to check the optimal destaining time before MS analyses, with plasma samples at least 90 minutes (fig 9). If the samples are not fully destained the signal to noise in the spectra are reduced.

Plasma proteins were separated with 2-DE and first stained with Sypro Ruby. The gel was then destained with 25 mM ammonium bicarbonate/50 % acetonitrile buffer and the efficiency of the process checked after different time intervals. Finally, the proteins were restained with silver. A: Proteins more stained by Sypro compared to silver. B: Glycoproteins more stained by silver. The rectangle shows an area containing additional proteins detected by silver. Residual Sypro staining of proteins after destaining are indicated with rings.

#### **4.3 Protein identification with peptide mass fingerprinting using MALDI-TOF MS**

Peptide mass fingerprinting with MALDI-TOF MS is an excellent and robust technique for fast identification of plasma proteins after 2-DE (Lahm & Langen, 2000). However, several peptide peaks needs to be detected in the spectra with a high mass accuracy to avoid false positive results. There are several approaches to consider for improving the data from the analyses, such as digestion protocol, purification of peptides and choice of matrix.
