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Chapter 4 : Protein Stable Isotope Probing
Category: Environmental Microbiology; Applied and Industrial Microbiology
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The chapter on protein stable isotope probing (protein-SIP) focuses on cultivation conditions, extraction of proteins, different protein-separation techniques, methods of mass spectrometry (MS) analysis, and the calculation of stable isotope incorporation levels. The outlook includes the use of other isotopes than carbon, other molecule classes than proteins, and how protein stable isotope probing (protein-SIP) is integrated into metaproteomic approaches for the analysis of structure and function of microbial consortia. This chapter describes the method of protein-SIP by means of proof-of-principle experiments using Pseudomonas putida strain ML2 as the model organism and with fully labeled [13C]benzene or 15NH4 as a model substrate. For intact protein profiling (IPP), measurements were performed in the positive voltage polarity ion reflector mode. Mostly, recorded MS/ MS-spectra are of poor quality and mass-tags of only a few generated amino acid-ions can be defined and compared with the database. The measurements for shotgun mass mapping (SMM) were performed with external calibration using the peptide calibration standard II, which covered a mass range from m/z ~700 to 3,200. In a study, P. putida ML2, an aerobic, heterotrophic proteobacterium, was grown in mineral medium in the presence of [12C]benzene, fully labeled [13C]benzene, or [12C]benzene and labeled 15NH4Cl. In the stationary growth phase, cells were harvested, and subsequently the whole protein extracts were separated by 2-D gel electrophoresis. One of the most important challenges is the identification of peptides from samples with dynamic and variable labeling. Therefore, new algorithms for the analysis of fragmentation spectra and database search are needed.
Workflow of protein-SIP. Depending on your experimental design and which stable isotopes are used, the culture grows on the same conditions, except the heavy isotope labeled carbon source. After cell harvesting and protein extraction, the samples are processed in parallel in order to obtain comparable results. Proteins or peptides are separated and fractionated and may be analyzed by several tools and techniques (see Fig. 3 ). In proteomics, the analysis ends with an MS measurement. Mostly, this is done by matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI) MS, depending on the aim of investigation and complexity of measured samples.
Workflow of protein preparation for MS analysis. The simplest way is shotgun proteomics, in which a crude protein extract or intact cells treated by a protease such as trypsin is purified and desalted by small reverse-phase columns and further analyzed by MALDI-MS or ESI-MS. In contrast, the gel-based approach achieves the fractionation and separation of proteins by gel electrophoresis (1D-SDS-PAGE or 2-D electrophoresis) and further enzymatic in-gel digestion (e.g., also with trypsin) but also ends with MALDI- or ESI-MS measurements.
Theoretical background of the halfdecimal-place rule (HDPR) calculation method. Typical theoretical 12C and 13C isotope-resolved peptides (m/z 500, 1,000, and 1,500) are shown in the upper panel, where the monoisotopic isotopes with the exact calculated decimal place are depicted. In the upper right panel, the mass-shifted decimal places of the corresponding 13C peptides are underlined. This information was used to plot as a reference, around 90,000 peptides of M. tuberculosis, in order to receive reference slopes for 0 atom% and 100 atom% 13C incorporation by a robust linear regression algorithm. They served as references for partial 13C incorporation. ( Fetzer et al., 2010 )
Scheme of functional metaproteomics: from “omics” data to biological content. After cultivation, fractions of the samples can be taken for DNA/RNA analyses in order to get a better overview of the structural composition of the analyzed culture. Black arrows indicate the workflow for the labeled sample, and in parallel for the unlabeled (control) sample, the white arrows are used. The central part of the workflow is database comparison with genome sequence information. Raw data acquired from the MS are computationally processed and further used for protein identifications. In conjunction with the information of stable isotope incorporation into specific functional proteins, a reconstruction of the microbial culture can be built up with the information of functional classification (e.g., extracellular or intracellular), taxonomic structure, and metabolic activity.
Representative MALDI-TOF/TOF-MS-spectra of the benzene 1,2-dioxygenase beta protein subunit. Three different PMF-spectra from the same protein. Isotopologues of peptides are visible and in the insets the unlabeled peptide ITSDVSWSEN-PASR at m/z 1548.74 from the 12C-benzene culture (A), the labeled peptide at m/z 1565.69 from the benzene/15NH4 culture (B), and the labeled peptide at m/z 1612.93 from the 13C6-sbenzene culture (C) are shown that allow the calculation of the number of incorporated nitrogen and carbon atoms in this peptide.
MS/MS-spectra of the peptide ITSDVSWSENPASR. The corresponding y-ions in each spectrum are indicated with their mass, except for the y13-ion at m/z 1435.65, which is not determined. (A) The y1-ion of arginine at m/z 175.10 indicates the natural isotopic distribution of the chemical elements. (B) The y1-ion at m/z 179.08 and (C) at m/z 181.49 reveals a mass shift, which indicated an incorporation of nitrogen and carbon. (Adapted from Jehmlich et al., 2008a , with permission from Wiley InterScience.)
Workflow of the shotgun proteomic approach. P. putida ML2 was grown independently with natural and labeled substrates; IPP and SMM approaches were applied in order to detect and quantify the metabolic incorporation of heavy isotopes (15N and 13C) into peptides and proteins. (Adapted from Jehmlich et al., 2008a , with permission from Wiley InterScience.)
Mass spectrometry analysis of selected proteins from the artificial mixed culture composed of EbN1 and UFZ-1 grown on different substrates. (A) A detailed view into a PMF-spectrum of the chaperone protein dnaK (EbN1) SLGQFNLSDIPPAPR m/z 1611.86 from the mixed culture grown with 12C-toluene/gluconate as well as [13C7]toluene/gluconate (C). (B) A detailed view of the PMF-spectra of the heat shock protein HSP20 family (gene sequence homology to Pseudomonas stutzeri A1501) from the mixed culture grown with [12C]toluene/gluconate is displayed as well as the sample from a culture grown on [13C7]toluene/gluconate (D). There was no 13C incorporation in peptides detectable belonging to the enrichment culture UFZ-1. (Adapted from Jehmlich et al., 2008b .)
Stable isotope composition of elements potentially relevant for protein-SIP