Multidimensional Protein Identification Technology-Selected Reaction Monitoring Improving Detection and Quantification for Protein Biomarker Studies

The targeted analysis of proteins in complex biological samples is best achieved using selected reaction monitoring (SRM). To maximize the sensitivity of this approach, sample fractionation or enrichment is still required, particularly to detect less abundant proteins in clinically relevant biofluids. Here, we report the development of multidimensional protein identification technology (MudPIT)-SRM, taking advantage of the robust online strong cation exchange chromatography for tryptic peptide fractionation and combining it with the multiplexed, quantitative attributes of SRM. The classical MudPIT method has been modified with an in-line strategy to introduce reference peptides onto the analytical column to enable quantitation at each salt step. Applying the MudPIT-SRM approach to profile abundant plasma proteins, we demonstrated mean increases in peak areas of almost 90% compared to conventional SRM. MudPIT-SRM analyses of low abundant proteins present in human wound fluid exudates similarly demonstrated increased peak areas and enabled the detection of proteins which were below the lower limit of detection when analyzed by conventional SRM. The MudPIT-SRM method is relatively facile to conduct and offers performance advantages to enhance sensitivity for biomarker studies

<i>Pseudomonas aeruginosa</i> Proteome under Hypoxic Stress Conditions Mimicking the Cystic Fibrosis Lung

Pseudomonas aeruginosa is a ubiquitous Gram-negative pathogen known to inhabit hypoxic mucus plugs of cystic fibrosis (CF) patient lungs. Despite the high prevalence and related patient mortality, the protein machinery enabling the bacterium to adapt to low oxygen environment remains to be fully elucidated. We investigated this by performing both SWATH mass spectrometry and data-dependent SPS-MS3 of TMT-labeled peptides to profile the proteomes of two P. aeruginosa CF isolates, PASS2 and PASS3, and a laboratory reference strain, PAO1, grown under hypoxic stress (O₂ < 1%) in media that mimic the nutrient components of the CF lung. Quantitated across all three strains were 3967 P. aeruginosa proteins, reflecting approximately 71% of predicted ORFs in PAO1 and representing the most comprehensive proteome of clinically relevant P. aeruginosa to date. Comparative analysis revealed 735, 640, and 364 proteins were altered by 2-fold or more when comparing low oxygen to aerobic growth in PAO1, PASS2, and PASS3, respectively. Strikingly, under hypoxic stress, all strains showed concurrent increased abundance of proteins required for both aerobic (<i>cbb</i>₃-1 and <i>cbb</i>₃-2 terminal oxidases) and anaerobic denitrification and arginine fermentation, with the two clinical isolates showing higher relative expression of proteins in these pathways. Additionally, functional annotation revealed that clinical strains portray a unique expression profile of replication, membrane biogenesis, and virulence proteins during hypoxia which may endow these bacteria with a survival advantage. These protein profiles illuminate the diversity of P. aeruginosa mechanisms to adapt to low oxygen and shows that CF isolates initiate a robust molecular response to persist under these conditions

PRO40 Is a Scaffold Protein of the Cell Wall Integrity Pathway, Linking the MAP Kinase Module to the Upstream Activator Protein Kinase C

<div><p>Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus <i>Sordaria macrospora</i>. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated <i>mik1</i> gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.</p></div

PRO40 is required for correct signaling via the CWI pathway.

<p>(A) Time course of MAK1 phosphorylation in <i>pro40</i> deletion (Δ; S69656) and overexpression strains (OE; T184.2NS11) in comparison to wildtype. Strains were grown for three to six days, and phosphorylated MAK1 was detected in a Western blot using an anti-phospho-p44/42 antibody. The signal for tubulin was used as internal standard. Representative immunoblots of two to four independent experiments with three technical replicates are shown. (B) Stress-induced MAK1 phosphorylation in <i>pro40</i> deletion (Δ; S69656) and overexpression strains (OE; T184.2NS11) in comparison to wildtype. Strains were grown for three days and subjected to 0.01% H₂O₂ for 0, 15, 30, and 45 minutes prior to harvesting. Phosphorylated MAK1 was detected using an anti-phospho-p44/42 antibody, and the signal for tubulin was used as internal standard. Representative immunoblots of three independent experiments with three technical replicates are shown. (C) Model of the scaffolding function of PRO40 for the CWI pathway. Details are discussed in the text.</p

Shared interaction network of MEK1 and PRO40.

<p>Venn diagram comparing the three datasets generated from affinity purification and mass spectrometry with strains Δpro40::PRO40-FLAG (PRO40), Δmek1::NTAP-MEK1 (MEK1), and Δpro40::NTAP-MEK1 (MEK1(Δpro40)). The 12 proteins found in all three datasets and the 17 proteins found only in the PRO40 and MEK1 datasets are represented by boxes with <i>S. macrospora</i> locus tag numbers or protein designations. The magenta and blue box color indicates that the transcript of the encoding gene belongs to the top500 transcripts (with respect to read counts) in protoperithecia and vegetative hyphae, respectively (data taken from a previous transcriptomics analysis <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004582#pgen.1004582-Teichert1" target="_blank">[41]</a>).</p

Phenotypic characterization of kinase deletion strains.

<p>(A) Sexual development was assayed after 7 days of growth on BMM slides. Mature perithecia are only generated in wildtype (wt) and complemented deletion strains (Δmik1::<i>mik1</i>, Δmek1::<i>mek1</i>, and Δmak1::<i>mak1</i>). Δmik1, Δmek1, and Δmak1 generate only protoperithecia. White scale bar, 100 µm; black scale bar, 20 µm. (B) DIC microscopy of hyphal fusion in subperipheral regions. Strains were grown on solid MMS with a cellophane layer for 2 days. Hyphal fusion bridges are indicated by black arrowheads, whereas hyphae that grow in close contact without fusion are indicated by white arrowheads. Scale bar, 10 µm.</p

Shared interaction partners of MEK1 and PRO40.

<p>#s, number of spectral counts; #p, number of peptide counts; cov, coverage (%). <i>Sm</i>, <i>S. macrospora</i>.</p><p>Shared interaction partners of MEK1 and PRO40.</p

Localization of MIK1, MEK1, and MAK1.

<p>(A) GFP-labeled MIK1 and MEK1 are present in the cytoplasm and absent from nuclei. GFP-labeled MAK1 localizes to the cytoplasm, but is also targeted to the nucleus. (B) Co-localization experiments with MEK1-GFP and H2B-tdTomato show that spherical organelles devoid of MEK1 labeling are nuclei (arrowheads). Scale bar, 10 µm.</p

The Δmek1/pro40 double mutants shares phenotypic characteristics with Δmek1 and Δpro40.

<p>(A) Sexual development was assayed after 7 days of growth on BMM slides. Δpro40 and the Δmek1/pro40 double mutant generate only protoperithecia. White scale bar, 100 µm; black scale bar, 20 µm. (B) Δpro40 and the Δmek1/pro40 double mutant are unable to undergo hyphal fusion, although hyphae often grow in close contact (white arrowheads). Scale bar, 10 µm. (C) Localization of GFP-tagged MIK1, MEK1, and MAK1 in vegetative hyphae of the pro40 mutant and Δpro40. Scale bar, 10 µm. (D) Localization of GFP-tagged MIK1, MEK1, and MAK1 in three days old protoperithecia of the wildtype, the pro40 mutant, and the <i>pro40</i> deletion strain Δpro40. Scale bar, 20 µm.</p

Interactions of PRO40 with components of the CWI pathway.

<p>(A) Structures of proteins used for yeast two-hybrid analysis. Derivatives generated in addition to full-length constructs are shown below the protein structures. (B) Yeast two-hybrid analysis of CWI pathway components and PRO40. Yeast cells were drop-plated on SD medium lacking leucine, tryptophan, histidine, and adenine. Empty squares indicate that interactions were not tested. (C) Schematic overview of signal transduction and protein-protein interactions within the PRO40-CWI complex. Signaling through the pathway is depicted by gray arrows; interactions are depicted by black arrows. (D) Interaction sites between PRO40, PKC1, MIK1, MEK1, and MAK1. Black bars represent interaction sites tested in yeast two-hybrid analyses (A, B). For reasons of clarity, only PRO40 is depicted as homodimer.</p