Quantitation of the ribosomal protein autoregulatory network using mass spectrometry

Abstract

Relative levels of ribosomal proteins were quantified in crude cell lysates using mass spectrometry. A method for quantifying cellular protein levels using macromolecular standards is presented that does not require complex sample separation, identification of high-responding peptides, affinity purification, or postgrowth modifications. Perturbations in ribosomal protein levels by overexpression of individual proteins correlate to known autoregulatory mechanisms and extend the network of ribosomal protein regulation.

Figure 1

LC/MS data. a) A low-resolution contour plot of a portion of the mass spectrum of crude E. coli cell lysate spiked with ¹⁵N-labeled 70S ribosomes. The entire mass spectrum is shown in Supplementary Figure S1. The isotopic envelope consisting of the monoisotopic peak and isotopomers is not resolved in this representation. Solid red boxes indicate pairs of peaks representing unlabeled and ¹⁵N-labeled pairs of peptides from ribosomal proteins. Dashed red boxes indicate unpaired peaks from non-ribosomal proteins. The peak pair for ribosomal protein L24, residues 34-43 (VIVEGINLVK, +2) is highlighted. b) A 1D mass spectrum generated by summing in the retention time domain (total width of 0.2 minutes). The spectrum shown here arises from protein L24 residues 34-43, also depicted in the low-resolution contour plot in (a). Data points are indicated by grey dots while the blue and orange lines represent the theoretical distributions of the unlabeled (sample) and ¹⁵N-labeled (standard) distributions respectively. These distrbutions are fit to the data using LS-FTC, with a final unlabeled:labeled or sample:standard ratio of 1.08. The peak just below 548 m/z units arises from imperfect ¹⁵N-labeling of the 70S ribosome standard (99.3%), and is useful in discriminating between peaks from the standard and those from the lysate that happen to have a similar m/z value.

Figure 2

Ribosomal protein level measurements from E. coli containing an empty plasmid. a) Initial relative protein levels compared to the ¹⁵N standard. Circles represent individual measurements from different peptides or charge states of the same peptide. b) The same data as in (a), represented as an average value with error bars indicating the standard deviation of measurements. The data is uniformly scaled so that the average values for the reference proteins S4 and L3 are set to 1. c) The same data as in (b), divided on a per-protein basis by the uniformly scaled values from the wild type reference sample. Values now indicate the relative cellular protein level compared to wild type. Errors were propagated from both the sample and reference datasets.

Figure 3

Relative cellular protein levels. Dashed lines are drawn at the mean of 1.025 as well as the cutoffs of 1.325, 0.725 and 0.375 for significantly increased, significantly decreased and highly decreased respectively. Values greater than 1.325 are colored green, values less than 0.725 are colored orange and values less than 0.375 are colored red. All other values are colored blue. Values outside the range of the plots are indicated by green arrows. Error bars are the standard deviation of measurements from multiple ¹⁴N/¹⁵N feature pairs corresponding to different ions (peptides and charge states) and include error due to normalization by the wild type reference. a) Protein levels for the S4 overexpression experiment. b) Protein levels for the S7 overexpression experiment. c) Protein levels for the S8 overexpression experiment. d) Protein levels for the S19 overexpression experiment.

Figure 4

The network of interactions observed as a result of ribosomal protein overexpression. Ribosomal protein genes are organized by operon, and listed by protein name. Other proteins are listed by gene name. Operon names are indicated on the left edge of the operon. Proteins which are overexpressed but for which no significant effects were observed are shaded in yellow, while those for which effects are observed are shaded in blue. Operons that were neither the source nor target of regulatory effects are omitted. The green arrows indicate an increased protein level as a result of overexpression while the red and black arrows indicate a decreased protein level as a result of overexpression. Black arrows are those effects that have also been previously observed. Thin arrows indicate significantly decreased (< 0.725) or increased (> 1.325) while thick arrows indicate highly decreased (< 0.375).

Figure 5

Sucrose gradients of crude cell lysate. a) Wild type E. coli. b) E. coli overexpressing ribosomal protein S4. Relatively reduced levels of 70S ribosomes and increased levels of 30S and 50S subunits are visible. Two small new peaks appear compared to the wild type gradient, one sedimenting before each of the two subunits. Presumably these are the result of accumulation of low levels of ribosomal subunit assembly intermediates.