Hybrid Data Acquisition and Processing Strategies with Increased Throughput and Selectivity: pSMART Analysis for Global Qualitative and Quantitative Analysis

Data-dependent acquisition (DDA) and data-independent acquisition strategies (DIA) have both resulted in improved understanding of proteomics samples. Both strategies have advantages and disadvantages that are well-published, where DDA is typically applied for deep discovery and DIA may be used to create sample records. In this paper, we present a hybrid data acquisition and processing strategy (pSMART) that combines the strengths of both techniques and provides significant benefits for qualitative and quantitative peptide analysis. The performance of pSMART is compared to published DIA strategies in an experiment that allows the objective assessment of DIA performance with respect to interrogation of previously acquired MS data. The results of this experiment demonstrate that pSMART creates fewer decoy hits than a standard DIA strategy. Moreover, we show that pSMART is more selective, sensitive, and reproducible than either standard DIA or DDA strategies alone

Fusogenicity and Functionality of EnvG.

<p>(a) 10⁷ 293T cells were transfected with pEnvG or empty vector using Mirus Trans-IT 293 according to manufacturer's protocol. 48 h post-transfection, 293T cells were overlaid with 2×10⁶ CD4+CCR5+ GHOST cells. 48 h after overlay, cells were visualized under light microscope and images were captured. (b) 10⁶ CD4+CCR5+ GHOST cells were infected as above after pre-incubation with anti-VSV-G (Vi10) and/or anti-Env cocktail. After infection, Vi10 and/or anti-Env cocktail was included in the culture media. Eight hours post infection, cells were visualized under light microscope and images were captured.</p

Serum anti-Env IgG antibody responses.

<p>(a) Mean anti-Env endpoint titers (4 animals/group) against JR-FL foldon trimer were determined over the course of the 11 week experiment as described previously.(b) Week 6 endpoint titers for mice primed with pEnvG/pIL-12 (group 1/2/3, N = 12), IN rVSV-EnvG₄-G₆ (group 4/5, N = 8), or IM rVSV-EnvG₄-G₆ (group 6/7, N = 8). *p<0.05 compared to group 1/2/3. (c) Week 8 endpoint titers for all groups. (d) Week 11 endpoint titers for all groups. *p<0.05 compared to group 1. ^#p<0.05 compared to group 2. ⁺p<0.05 compared to group 3. ∧p<0.05 compared to group 4. ^∇p<0.05 compared to group 5. (e) Ratio of IgG2a to IgG1endpoint titers at week 11. (f) Neutralization of HIV-1 virus pseudo-typed with SF162.LS Env as measured in a standard TZM-bl neutralization assay using IgG purified from week 11 sera of selected groups. SEM is shown.</p

Genetic layout of the rVSV-EnvG₄-G₆ vector, HIV-1 Env, and the EnvG insert.

<p>VSV genes are shown in the 3′ to 5′ orientation as ordered on the recombinant genomic VSV plasmid and are not to scale. Arrows below each VSV gene depict the diminishing 3′-to-5′ mRNA transcription gradient. ss: signal sequence. MPER: membrane proximal external region. TM: transmembrane domain. CT: cytoplasmic tail domain. Star denotes site of intracellular Env(G) cleavage by furin.</p

Frequency of JR-FL Env-specific CD4+ T cell responses in the lung and spleen.

<p>1.5×10⁶ leukocytes isolated from the lungs (a, b) andspleen (c, d) at the end of the study were stimulated <i>ex vivo</i> with JR-FL gp140, anti-CD28 and brefeldin A before being analyzed by flow cytometry for production of IFNg, IL-2 and TNFa. The total frequency of cytokine secreting CD4+ T cells (%) producing IFNγ, IL-2 or TNFα are shown on the left (a, c) and the frequency of multifunctional CD4+ T cells producing any combination of IFN γ +, IL-2+, and/or TNFα+ on the right (b, d). *p<0.05 compared to group 1. ^#p<0.05 compared to group 2. ∧p<0.05 compared to group 4. ^∇p<0.05 compared to group 5. ^@p<0.05 compared to group 6. ^xp<0.05 compared to group 7. Bars represent the median (with SEM), individual animals are shown.</p

rVSV-EnvG₄-G₆ characterization.

<p>(a) After a 24 hr infection, total infected Vero cell lysates were collected and proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE). Blot was probed with anti-VSV-N and anti-VSV-G_IN CT, which does not recognize G_NJ. IN: rVSV-EnvG₄-G₆^IN; NJ: rVSV-EnvG₄-G₆^NJ. (b-d) Sucrose-gradient purified rVSV-EnvG₄-G₆ particles were separated by SDS-PAGE. (b) Blots of 10⁶ pfu of rVSV-EnvG₄-G₆^IN and 2.5×10⁶ pfu rVSV-EnvG₄-G₆^NJ vectors were probed separately for EnvG using anti-gp120. (c) Blot of 10⁶ pfu rVSV-EnvG₄-G₆^IN was probed with anti-VSV-G^IN CT. (d) Blot of 10⁶ pfu rVSV-EnvG₄-G₆^IN and rVSV-EnvG₄-G₆^NJ was probed with anti-VSV-G. (e) Replication kinetics of rVSV-EnvG₄-G₆ and rVSV-G₄ viruses in Vero cells. Vero cells were infected in 6-well plates at an MOI of 0.1. At various intervals post infection, supernatant was collected from duplicate wells and virus was titrated. Plaques were counted manually after cell fixation.</p

Effect of IN rVSV-EnvG₄-G₆ adminstration.

<p>(a) Mean body weights and temperatures (b) of inoculated mice. (c) Mean copy numbers of VSV N genomic RNA or (d) VSV N mRNA per mg of indicated tissue or mL of blood. N = 4–12, dependent on study day. Fresh tissue specimens were homogenized, clarified by centrifugation and supernatants were subjected to RNA extraction and qPCR. All samples were tested in duplicate. Dotted lines indicate limits of detection. SEM is shown. *<i>p</i><0.05 for comparison of rVSV-EnvG₄-G₆ to rVSV-G₄. All PBS and rVSV-EnvG₄-ΔG values were found to be significantly lower than rVSV-EnvG₄-G₆ and rVSV-G₄ values.</p

Murine Immunization Regimens.

<p>* Groups 1–3 were primed twice, at weeks 0 and 3; groups 4–7 were primed once at week 3.</p><p>∧All groups were boosted at week 6. <b>IN</b>: Intranasal; <b>IM</b>: Intramuscular. <i>Superscript</i> IN/NJ denotes strain of rVSV used.</p><p>Murine Immunization Regimens.</p

Surface staining of rVSV-EnvG₄-G₆-infected Vero cells and rVSV-EnvG₄-G₆ particles.

<p>(a) After a 22 hr infection, Vero cells were detached from plate by gentle trypsin treatment and resuspended in PBS. 5×10⁶ cells were analyzed for VSV G and HIV-1 EnvG surface expression. All cells analyzed for Env staining were first gated as positive for G staining. (b) For rVSV staining, 10⁹ pfu of virus was bound to alum at 37°C with agitation. rVSV/alum conjugates were stained with titrated anti-VSV-G (Vi10) or anti-HIV-1 Env Ab followed by anti-human IgG or anti-mouse IgG2a Alexa555 and acquired on a modified LSRII flow cytometer. Median fluorescent intensity (MFI) was determined for each Ab dilution. (c) 10⁹ pfu of virus was incubated with SYTO 63 nucleotide stain in PBS for 30 min at RT followed by incubation with anti-VSV-G (Vi10) and then anti-mouse IgG2a Alexa555. Virus was analyzed as described above. Minimum threshold settings on SSC were used to increase sensitivity for small particles and FSC and SSC parameters were set to log scale. Deionized water was run for 15 min to equilibrate for low threshold noise. ∼50,000 events were acquired for the PBS control, ∼10⁶ events were acquired for virus samples. Particles staining positive for nucleic acid that were above the noise threshold were gated on, and the amount of anti-G staining for those populations were compared.</p