Effective coupling of CE with nanoESI MS via a true sheathless metal-coated emitter interface for robust and high sensitivity sample quantification (ASMS 2016)

<p>Capillary electrophoresis (CE) coupled with mass spectrometry (MS) is a promising alternative to conventional liquid chromatography (LC) MS in chemical and biological sample analysis due to its high resolving power and fast separation speed. Reproducibility and ruggedness problems, suffered to a certain degree by almost all the CE-MS interfaces, limit its broad applications. We present the development of a new sheathless CE-MS interface aiming at overcoming these problems and pushing CE-MS suitable to routine sample analysis with high sensitivity. A systematic evaluation of the new interface was performed using a hybrid capillary isotachophoresis (CITP) and capillary zone electrophoresis (CZE) separation coupled with electrospray ionization (ESI) MS for its achievable sensitivity and reproducibility in sample quantification. </p

Common proteins significantly altered by injury and by NSAID administration.

<p>Common proteins significantly altered by injury and by NSAID administration.</p

IPA predicts an NSAID-induced Increase in Cell Death and a Reduction in Proliferation/Survival in Regenerating TA Muscles.

<p>IPA predicts an NSAID-induced Increase in Cell Death and a Reduction in Proliferation/Survival in Regenerating TA Muscles.</p

NSAID-induced decrease in anti-apoptosis proteins is associated with increased caspase activity.

<p>Caspase 3/7activity in muscle homogenates from saline- and NSAID-treated animals (4/group) was measured at 54 hrs after injury (7 hrs post-treatment) by commercial ELISA. Data are given as the fold-change for each animal relative to its own non-injured TA muscle. Responses between saline versus NSAID-treated animals were statistically significant as determined by ANOVA using a mixed model with random effect of animal within treatment with the level of significance set at p < .05.</p

Experimental muscle injury disrupts myofiber architecture and causes marked influx of inflammatory cells.

<p>Mice underwent the eccentric contraction (EC) exercise regimen as described in Methods. At 24 hr post-EC, animals were euthanized and both left and right tibialis anterior (TA) muscles were harvested post-mortem for routine histopathology. One representative animal of 3 is depicted. The exercised muscle, but not the control TA, displays destruction of normal muscle architecture and a marked inflammatory cell influx. These pathologies, and the corresponding reduction in muscle force, are accepted criteria that define muscle injury.</p

AMT-tag proteomics identifies proteins from injured muscles that are significantly altered by NSAID treatment.

<p>(A) Proteins that met both the criteria of “Coverage” (defined as: [Number of paired injured and control muscles ≥ 50% or (N_(injured) or N_(control)) ≥75%]) and of “Difference” (defined as: [|Fold Change| ≥1.25 or |N_(injured)—N_(control)| ≥ 50%)]) were subjected to statistical analysis to identify those that were significantly affected by NSAID treatment. Proteins were analyzed for quantitative effects using a standard T-test and for qualitative effects using G-test methodology [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172486#pone.0172486.ref032" target="_blank">32</a>]. In total, 277 differentially expressed proteins were found to be significantly altered by NSAID administration. (B) Functional categories of the 277 differentially expressed proteins (assigned as described in Methods; Table in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172486#pone.0172486.s002" target="_blank">S2 Table</a>: Proteins Significantly Altered by NSAID) are depicted. PTM: post-translational modification; ECM: extracellular matrix.</p

Experimental eccentric contraction injury stimulates physiological responses characteristic of muscle regeneration.

<p>Mice (4-12/group) underwent the EC regimen described above. Non-injured and injured TA muscles were harvested at the indicated times post-injury, flash-frozen and used to evaluate the relative expression of genes involved in muscle regeneration by qRT-PCR as described in Methods. Data are given as the mean fold-change in gene expression relative to the non-injured muscle ± standard error of the mean (SEM). Calculation of the 95% confidence interval using the log₂-transformed fold-change values revealed that all genes were significantly increased at each time point relative to the non-injured control as indicated by the grouped asterisks, except the 24h <i>ptgs1</i> expression. No corresponding proteomics analyses were conducted at 24 or 48 hrs; at 54 hrs post-injury the only corresponding protein that was significantly altered by injury was vimentin (Table in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172486#pone.0172486.s001" target="_blank">S1 Table</a>: Proteins Significantly Altered by Injury; 1.745-fold increase; <i>p</i> = .0236).</p

Deep-Dive Targeted Quantification for Ultrasensitive Analysis of Proteins in Nondepleted Human Blood Plasma/Serum and Tissues

Mass spectrometry-based targeted proteomics (e.g., selected reaction monitoring, SRM) is emerging as an attractive alternative to immunoassays for protein quantification. Recently we have made significant progress in SRM sensitivity for enabling quantification of low nanograms per milliliter to sub-naograms per milliliter level proteins in nondepleted human blood plasma/serum without affinity enrichment. However, precise quantification of extremely low abundance proteins (e.g., ≤ 100 pg/mL in blood plasma/serum) using targeted proteomics approaches still remains challenging, especially for these samples without available antibodies for enrichment. To address this need, we have developed an antibody-independent deep-dive SRM (DD-SRM) approach that capitalizes on multidimensional high-resolution reversed-phase liquid chromatography (LC) separation for target peptide separation and enrichment combined with precise selection of target peptide fractions of interest, significantly improving SRM sensitivity by ∼5 orders of magnitude when compared to conventional LC-SRM. Application of DD-SRM to human serum and tissue provides precise quantification of endogenous proteins at the ∼10 pg/mL level in nondepleted serum and at <10 copies per cell level in tissue. Thus, DD-SRM holds great promise for precisely measuring extremely low abundance proteins or protein modifications, especially when high-quality antibodies are not available

A carrier-assisted targeted mass spectrometry approach for proteomics analysis of single cells (ASMS 2017)

Antibody-based flow cytometry and mass cytometry are predominant technologies for targeted proteomics analysis of single cells. However, they share common shortcomings with other antibody-based methods (e.g., low-multiplex, the need of high-quality antibodies, and unavailability of antibodies for new proteins). Furthermore, they lack quantitation accuracy to provide accurate protein concentrations. Mass spectrometry (MS)-based targeted proteomics has emerged as a promising alternative for antibody-free precise high-multiplex quantification of target proteins. However, it does not meet the merit for analysis of single cells due to insufficient sensitivity. To tackle this issue, we recently developed a new targeted MS approach that couples carrier-assisted sample preparation to a highly sensitive targeted MS platform for enabling proteomics analysis of single cells. In complex biological samples low abundant proteins can be reliably detected without loss because highly abundant proteins severe as an effective carrier to prevent their loss. With this observation, we recently developed a simple preparation method with using exogenous BSA protein as a carrier for lossless processing of single cells. A highly sensitive targeted MS platform PRISM-SRM/PRM (high-pressure, high-resolution separations with intelligent selection and multiplexing coupled to selected/parallel reaction monitoring) was then used for absolute quantification of key EGFR pathway proteins in 1-100 HMEC cells. The Waters nanoACQUITY UPLC system and the Thermo Scientific TSQ Vantage or Q Exactive MS instrument were used for LC separation and targeted quantification, respectively. The Skyline software was employed for analysis of SRM/PRM data.Recently PRISM-SRM was demonstrated for enabling highly sensitive quantification of target proteins at ≤100 copies per human cell when starting with only ~25 µg of cell lysate digests, which is equal to ~250,000 human cells. However, in the real clinical applications the available sample amount could be much smaller (e.g., 1-10 circulating tumor cells) and sample loss is almost inevitable for current targeted MS analysis. With the introduction of exogenous proteins as a carrier to prevent sample loss we refined our PRISM termed carrier-assisted PRISM (i.e., cPRISM) for enabling targeted quantification of proteins in single cells by coupling with SRM/PRM. In our proof-of-concept experiment BSA protein was selected as a carrier for assisting processing of a small number of cells. 1-100 isolated HMEC cells were collected into 50 µg of BSA carrier-containing tubes to prevent cell adhesion to the tube wall. When mixed with BSA carrier, the small number of cells were processed as easily as bulk cells with minimal sample loss. The constant ‘unbiased’ peptide recovery was observed across all the samples with confident detection of multiple endogenous peptides with different hydrophobicity at 1-5 HMEC cells as well as the linear response curves of target proteins from 1 to 100 cells. This result has shown the carrier-assisted method can be used to effectively process single cells for highly sensitive PRISM-SRM quantification. Furthermore, detection of SFADINLYR derived from NRAS in small numbers of HMEC cells (~80,000 NRAS molecules per HMEC cell from bulk cells) suggested that our current targeted MS platforms can provided ~100 zmol level of sensitivity. Currently we are working on further improving cPRISM-SRM performance in sensitivity, throughput and robustness. We envision that this new targeted MS approach will have broad utilities in biomedical research (e.g., single cells and clinical applications with extremely small sample amounts)

Peak areas of individual measurements

This data contains peak areas of individual measurements of the light (or endogenous) and heavy peptides from the 5 experiments.<div>a: Experiment 1_Batch 1</div><div>b: Experiment 1_Batch 2</div><div>c: Experiment 2<br></div><div>d: Experiment 3<br></div><div>e: Experiment 4<br></div><div>f: Experiment 5<br></div