Segmentation of Precursor Mass Range Using “Tiling” Approach Increases Peptide Identifications for MS¹‑Based Label-Free Quantification

Label-free quantification is a powerful tool for the measurement of protein abundances by mass spectrometric methods. To maximize quantifiable identifications, MS¹-based methods must balance the collection of survey scans and fragmentation spectra while maintaining reproducible extracted ion chromatograms (XIC). Here we present a method which increases the depth of proteome coverage over replicate data-dependent experiments without the requirement of additional instrument time or sample prefractionation. Sampling depth is increased by restricting precursor selection to a fraction of the full MS¹ mass range for each replicate; collectively, the <i>m</i>/<i>z</i> segments of all replicates encompass the full MS¹ range. Although selection windows are narrowed, full MS¹ spectra are obtained throughout the method, enabling the collection of full mass range MS¹ chromatograms such that label-free quantitation can be performed for any peptide in any experiment. We term this approach “binning” or “tiling” depending on the type of <i>m</i>/<i>z</i> window utilized. By combining the data obtained from each segment, we find that this approach increases the number of quantifiable yeast peptides and proteins by 31% and 52%, respectively, when compared to normal data-dependent experiments performed in replicate

Relative Frequencies of Epitope Occurrences in the Genome and CD8⁺ T-Cell Responses

<div><p>(A) Venn diagram representing relative frequencies and overlap of T cells responding to T. cruzi epitopes. The blue shaded circles (left) show the high-abundance overlapping peptides TSKB21 and TSKB20, along with the low-abundance overlapping peptide TSKB260. These peptides are among the most highly represented H-2K^b–binding peptides in the proteome and elicit the strongest CD8⁺ T-cell response following infection with Brazil strain <i>T. cruzi,</i> as shown in (B). The smaller purple shaded circles (right) show the lower-abundance epitopes TSKB18, TSKB74, TSKB80, and TSKB89, which are recognized by T-cell populations that overlap minimally with TSKB20/21/260-specific T cells.</p><p>(B) Relative frequencies of CD8⁺ T cells recognizing the TSKB20 (blue) and TSKB18 (purple) families of peptides in the acute (left) and chronic (right) phase of T. cruzi infection in mice infected with three different strains. While there are strain-dependent differences in the magnitude of the TSKB20-specific T-cell responses, these responses all follow a similar pattern of expansion and contraction during acute infection, which may be explained simply by strain-dependent differences in the rates of parasite replication and/or expression levels of TSKB20. However, T-cell responses to TSKB18 vary remarkably in both their magnitude and kinetics following infection with the various strains. Sustained expansion of TSKB18-specific T cells following infection with CL or Y strains results in different hierarchies of Ag-specific CD8⁺ T cells in chronic infection with the three different strains (right).</p></div

Parasite Strain–Dependent Dominance Patterns in T. cruzi–Infected Mice

<p>B6 mice were infected with 1,000 Brazil, 1,000 Y, or 10,000 CL strain T. cruzi and stained with TSKB20/K^b (top) or TSKB18/K^b (bottom) tetramers as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020077#s4" target="_blank">Materials and Methods</a>. Data shown are a combination of two separate experiments with three or four mice per group per experiment.</p

Detection of CD8⁺ T Lymphocytes Recognizing HLA-A2.1–Restricted ts Epitopes in Patients with Chronic Chagas Disease

<p>PBMCs were stained using MHC tetramers specific for the TSA2–38 or TSA2–44 epitopes. The percentage of CD8⁺ T cells staining positive for TSA2–38 is shown for a representative chronic chagasic patient and an uninfected control. Cells shown are lymphocytes gated on CD4^negB220^negCD11b^neg lymphocytes. Five out of eight IFNγ ELISPOT⁺ subjects stained positive with tetramers.</p

Functional Responses of Murine CD8⁺ T Cells Directed against Non-ts Epitopes

<div><p>(A) SCs from naive (gray bars) or chronically infected (Brazil strain; black bars) B6 mice were stimulated for 5 h with TSKB20, cruzipain, or GFT peptides plus GolgiPlug as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020077#s4" target="_blank">Materials and Methods</a>. Data represent the number of CD8⁺ T cells producing IFNγ; error bars represent SD.</p><p>(B) SCs from naive B6 mice were loaded with T. cruzi peptides and labeled with CFSE as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020077#s4" target="_blank">Materials and Methods</a>. The numbers above the peaks represent the percentage of specific killing of the target cells loaded with the indicated peptide and was calculated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020077#s4" target="_blank">Materials and Methods</a>. Data are representative of three experiments.</p></div

Magnitude, Kinetics, and Cross-Reactivity of CD8⁺ T-Cell Responses to Dominant ts Epitopes

<div><p>(A) SCs from naive or chronically infected (Brazil) B6 mice were stained with TSKB20/K^b-PE tetramers, then anti-CD8 APCs and a Cy5PE exclusion channel consisting of anti-CD4, anti-CD11b, and anti-B220. Cells shown are gated on Cy5PE^neglymphocyte⁺ populations. Numbers represent the percentage of CD8⁺ T cells staining with the TSKB20/K^b tetramer.</p><p>(B) Kinetics of TSKB20 (closed triangles)– and TSKB18 (open triangles)–specific responses during Brazil strain infection of B6 mice. Data are representative of three experiments (<i>n</i> = 5 mice per group).</p><p>(C) SCs from infected mice were stained with anti-CD8–FITC, TSKB20/K^b-PE, anti-CD4/11b/B220 cocktail, and either TSKB18/K^b–APC (top) or TSKB21/K^b–APC (bottom). Left panel shows CD8 versus TSKB20/K^b; middle panel shows CD8 versus TSKB18/K^b or TSKB21/K^b; right panel shows TSKB18/K^b or TSKB21/K^b tetramer staining versus TSKB20/K^b staining, gated on CD8⁺ lymphocytes.</p></div