Methodology And Strategy

One of the central points in any proteomics study is the preparation of the biological samples that are analyzed. Although current biomolecular MS enables very sophisticated applications such as detection of low-abundance proteins in complex mixtures, there are limits for the sensitivity of detection and the dynamic range that need to be considered in the design of a proteomics study. The results of the MS analysis in the end of a work flow cannot be better than the samples that are applied for it. For this reason we focus on the preparation of samples in different chloroplast pro-teome studies. We also add a brief discussion of the analytical approaches to identify the proteins of the different chloroplast fractions. However, details of conventional proteomics techniques are presented elsewhere in this book.

The experimental design of most proteomics studies includes three steps that contain (i) a sample preparation and (ii) 1D or 2D separation by electrophoresis or chromatography, followed by (iii) MS analysis of the separated proteins. While the last two steps usually involve standard techniques, the first step does not. Any sample preparation starts with the choice of the plant material that is critical for the outcome of a proteome study and its reproducibility. The majority of studies used soil-grown Arabidopsis plants. However, plants grown hydroponically or on plates were also used. The age of the plants varied from 3 to 4 up to 7 weeks. With respect to light and illumination times, the variations are somewhat smaller. Normally the light intensity was 100-200 |mol photons m-2 s-1 with 150 |mol photons m-2 s-1 in most cases; illumination times were between 8 and 14 h. So far no studies have been conducted on how these parameters affect the chloroplast proteome. The broad range of growth conditions used in different studies suggests that the proteome profiles display snapshots that show parts of the chloroplast proteome at different times of plant development. Figure 23.2 gives an overview of principal steps that were used in the sample preparation of some important studies of the chloroplast proteome. In many studies, Percoll gradient centrifugation was used to purify intact chloroplasts from broken chloroplasts and membranes from other organelles [1, 10, 11, 13-15]. The study of the luminal chloroplast proteome used chloroplasts that were purified by differential centrifugation to prepare a high amount of start material for a further purification of the luminal proteins [2]. To enable identification of low-abundance proteins, the sample preparation included usually the purification of a subcellular

Lumen

Broken chloroplasts (thylakoids)

Yeda press rupture 2D-PAGE -> MALDI-TOF-MS, Edman 50 proteins (36 lumenal) [2]

Thylakoids

Broken chloroplasts (thylakoids)

Yeda press rupture 2D-PAGE -> MALDI-TOF-MS, Edman, ESI-MS/MS 76 proteins (30 lumenal) [3] / Intact chloroplasts

KBr/KNO3 wash Extraction (Butanol, (NH^2SO4, pH) Gel-C-MS/MS 242 proteins [8]

Extraction (Na2CO3,Triton X-114, chloroform/methanol) 2D-PAGE, MALDI-TOF-MS Gel-C-MS/MS, ESI-MS/MS 154 proteins [7]

Yeda press rupture 2D-PAGE -> MALDI-TOF-MS, Edman, ESI-MS/MS 76 proteins (30 lumenal) [3] / Intact chloroplasts

Extraction (Na2CO3,Triton X-114, chloroform/methanol) 2D-PAGE, MALDI-TOF-MS Gel-C-MS/MS, ESI-MS/MS 154 proteins [7]

Envelope

Sucrose gradient Sucrose gradient

Extraction (NaOH or NaCl, Gel-C-MS/MS or chloroform/methanol) Off-line MUDPIT & Gel-C-MS/MS LC-MS/MS

54, and 112 proteins [10,11] 392 proteins [12]

Triton-insoluble fraction

2 x 1 % Triton X-100 extraction, insoluble proteins analysed by Gel-C-MS/MS 179 proteins [14]

Percoll gradient centrifugation

Percoll gradient centrifugation

Stroma

Hypotonic lysis in Hepes Native gel/SDS-PAGE -> MALDI-TOF-MS, LC-MS/MS 241 proteins [9]

Whole chloroplast

Percoll gradient repeated 3 x Extraction (Tris, Urea,Chaps/Brij, SDS) Chromatography (ion excahnge and Blue sepharose) Gel-C-MS/MS

690 proteins (636 chloroplast proteins) [1]

Envelope

Sucrose gradient Sucrose gradient

Extraction (NaOH or NaCl, Gel-C-MS/MS or chloroform/methanol) Off-line MUDPIT & Gel-C-MS/MS LC-MS/MS

54, and 112 proteins [10,11] 392 proteins [12]

Triton-insoluble fraction

Hypotonic lysis in Na-pyrophosphate 2D-PAGE, DIGE analysis of cold response -> MALDI-TOF-MS 43 differentially displayed proteins [26]

2 x 1 % Triton X-100 extraction, insoluble proteins analysed by Gel-C-MS/MS 179 proteins [14]

Plastoglobuli

Vidi et al. 2006, and Ytterberg et al. 2006

Sucrose gradient - ultracentrifugation

Gel-C-MS/MS

FIGURE 23.2. General methods used in chloroplast proteome studies.

compartment or a systematic fractionation of the organelle. For instance, envelope membranes and plastoglobules were purified using sucrose density centrifugation [10, 11, 15, 16]. Stromal chloroplast fractions were purified by classical hypotonic lysis [9, 26]. Thylakoid membranes were purified by different wash treatments [7, 8]. Rupture of carefully washed thylakoids using a Yeda press was an easy and efficient method to isolate a highly pure fraction of soluble lumen proteins [2]. A similar approach using differently purified thylakoids yielded a fraction of peripheral thylakoid proteins that contained a higher amount of stroma proteins [3].

Because the chloroplast is a membrane-rich organelle (Figure 23.1), specialized techniques were needed to extract the hydrophobic membrane proteins of the envelope and the thylakoid membrane. For instance, C/M solutions have strong interactions with lipids and were therefore useful to extract proteins from the hydrophobic core of the envelope [10]. In combination with alkaline and salt treatments, this method enabled identification of a broad range of the peripheral and integral envelope proteins [11]. A combination of salt and organic solvent treatments resulted also in an effective fractionation of thylakoid membrane proteins [7]. An alternative approach to fractionate the proteins of the thylakoid membrane involved a wash with chaotropic agents (KBR, KNO3) to remove a large part of the peripheral proteins. Following extraction of the pigments, the remaining peripheral and integral thylakoid proteins were then fractionated using sequential (NH4)2SO4 precipitation and pH shifts. This approach enabled identification of many known or potential integral thylakoid proteins [8]. The variety of different methods that have been used to extract proteins of different solubility from the envelope, and the thylakoid membrane showed that none of the extractions was complete. Instead a combination of complementary extraction steps had to be performed to cover a broad range of envelope and thylakoid proteins. As for the entire chloroplast, sequential extraction using four different buffer systems including Tris, urea, thiourea, and detergents provided suitable samples for a large-scale identification of chloroplast proteins [1]. An alternative approach removed the major part of the envelope and thylakoid membranes by solubilization with Triton X-100. The fraction of Triton X-100 insoluble proteins comprised a complementary set of chloroplast proteins, many of which had not been identified in the large-scale analysis of the entire chloroplast proteome [14].

An important part in the workflow of proteomics involves separation of the proteins by electrophoresis or chromatography before their analysis by MS. A classical approach combines 2-DGE and MALDI-TOF-MS or ESI-MS. However, this approach was only useful for studies of soluble proteins such as luminal and stromal chloroplast proteomes or peripheral thylakoid proteins [2, 3, 8, 26]. As for membrane proteins, separation by BN-PAGE combined with SDS-PAGE was a successful approach to study assembly and turnover of PS II under light and iron stress conditions [17, 18]. Alternatively membrane proteins were separated by RP chromatography before enzymatic cleavage of the collected protein fractions and their analysis by MS/MS [7, 8]. A common design to analyze membrane proteins involved a 1D separation by SDS-PAGE that was followed by enzymatic or chemical cleavage of the excised protein bands. The resulting peptides were further separated by nRP-HPLC and analyzed on-line by MS/MS (Gel-C-MS/MS). This approach was, for instance, applied in studies of the chloroplast envelope proteome [10-12], and it enabled the identification of more than 100 proteins in the different fractions of extracted envelope proteins of Arabidopsis [11, 12]. A complementary approach for the analysis of envelope proteins involved MudPIT. Instead of a separation of intact proteins, the purified envelope proteins were digested using trypsin and the peptides separated ion exchange chromatography. The collected peptide fractions were further separated by on-line RP-HPLC, and the eluted peptides were analyzed on-line by MS/MS. The combination of MudPIT with Gel-C-MS/MS enabled the identification of 392 proteins, 149 of which were only found by MudPIT [12]. A series of multidimensional separation steps was also used in the study of the entire chloroplast proteome. Separation of the extracted chloroplast proteins by ion exchange chromatography or Blue Sepharose affinity chromatography was combined with SDS-PAGE to resolve the complex mixtures of chloroplast proteins. Individual fractions and protein bands were cleaved by enzymatic digestion, and the resulting peptides were analyzed by RP-LC-MS/MS. This design enabled the identification of 690 chloroplast Arabidopsis proteins, 636 of which were considered to be part of the chloroplast proteome [1].

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