SIL-Assisted MS. SIL is commonly used in coupling of MS in proteomics studies, particularly for quantitative analysis of abundance of cellular proteins between different experimental samples. This SIL-assisted MS approach is widely employed to study differential protein expression related to signal transduction and gene regulation. The general strategy for any SIL-MS method is to induce mass shift by introducing a specific mass tag to cellular proteins and peptides. For quantitative analysis, the proteins in one sample are not tagged, but the proteins in another sample are tagged. Two samples are equally mixed together; MS measurement of the tagged and the nontagged peptide ions can provide a quantitative ratio for the same protein between two samples based on the measured signal intensities.
There are several ways for creating an SIL tag, either in vitro or in vivo . For example, 18O-labeled water can be used in trypsin digestion to introduce a 2-Da mass tag through the enzymatic reaction to uniformly label all proteolytic peptides at the C-terminus. Similarly, peptides can also be uniformly tagged at their N-terminus through the acetylation reaction, which introduces a 42-Da tag. The Cys-containing peptides can be selectively isolated and characterized using ICAT . The mass-coded abundance tagging (MCAT) can be used to characterize Lys-containing peptides for protein quantification . More recently, isobaric tags for relative and absolute quantitation (iTRAQ) technique has been used to uniformly label the N-terminus of the proteolytic peptides for multiplex quantitative comparison using several mass reporters in MS/MS spectrum .
Plant cellular proteins can also be mass-tagged through cellular metabolism in the process of protein translation. Metabolic labeling can be achieved using either 15N universal labeling  or SILAC . By using 15N-labeled nitrogen source in growth medium, all plant proteins are mass-tagged during translation. After trypsin digestion, the measured peptide ions will show a certain mass increase. However, the mass shift is not a fixed value because individual peptides may contain different number of nitrogen atoms dependent on their amino acid contents. Quantification using 15N-labeled peptides can be difficult due to this uncertainty. The SILAC strategy tags a particular type of amino acid residue with predictable mass shifts in MS spectra. However, since plants are autotrophic, labeled amino acids in the medium can be metabolized to other compounds after being uptaken into plant cells. Among several amino acids tested, only arginine showed relatively high labeling efficiency with approximately 80% of the labeled arginine incorporated into plant cellular proteins . Overall, the SIL-assisted MS approach has seen limited success in plants and has not been used for vacuolar proteins.
A Label-Free Strategy for Quantitative Proteomics. Since SIL methods need relatively expensive reagents and also require specific sample preparation procedures, an alternative label-free method has been proposed by several groups of researchers including us [28-31]. We believe that this label-free quantitative approach may be of particular value to plants.
A general method for the label-free approach is described as in the diagram (Figure 27.1). For the two samples that need be compared quantitatively, the LC-MS/MS experiment is first performed for both samples to determine protein constituents, and a precursor ions m/z and retention time file is generated for all MS/MS spectra of each identified protein. After the LC-MS/MS experiment, the LC-MS
Proteins digested with trypsin
Tryptic peptides 1 X \2
Extracting ion chromatograms
(quantification) -1- m/z
FIGURE 27.1. A schematic diagram to show a general strategy of the label-free quantitative proteomics.
experiment is immediately followed for the quantitative purpose. In the LC-MS spectrum, all peptide ions are individually extracted to retrieve total signal spectral counts using the same principle as for the labeling approach, and these spectral counts are used to quantify the relative difference of proteins in comparison between samples. Unlike the labeling methods with which quantitative analyses are limited to only the tagged peptides, with label-free methods essentially all peptides can be used and the quantitative comparison can be multiplex instead of binary. An example of applying this label-free method in study of an Arabidopsis mutant with VPE-depleted vacuoles will be described in the following section.
For the label-free quantitative approach, of most concern is (a) the variability associated with sample preparation and injection and (b) LC-MS experimental consistency. To normalize out these possible variables, spectral counts of several of trypsin's auto-cleavage peptides can be chosen for this purpose if the same amount of trypsin is used for different samples under comparison. For samples containing a large number of proteins, it is expected that only some proteins may be significantly different in abundance while the bulk of the proteins should be similar and can be used for normalization. When estimating change of PTM level of a particular peptide, the non-PTM peptides of the same protein should be chosen for normalization. Finally, researchers are searching for small nonretentive hydrophilic compounds that can be used for normalizing any fluctuations during entire LC-MS experiment. Of course, all of these analyses must require an automated program in order to carry out large-scale multiplex mathematical calculations.
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