Electron Capture vs Transfer Dissociation for Site Determination of Tryptic Peptide Tyrosine Sulfation: Direct Detection of Fibrinogen Sulfation Sites and Identification of Novel Isobaric Interferences.

Tyrosine sulfation, an understudied but crucial post-translational modification, cannot be directly detected in conventional nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) due to the extreme sulfate lability. Here, we report the detection of sulfate-retaining fragments from LC-electron capture dissociation (ECD) and nanoLC-electron transfer higher energy collision dissociation (EThcD). Sulfopeptide candidates were identified by Proteome Discoverer and MSFragger analysis of nanoLC-HCD MS/MS data and added to inclusion lists for LC-ECD or nanoLC-EThcD MS/MS. When this approach failed, targeted LC-ECD with fixed m/z isolation windows was performed. For the plasma protein fibrinogen, the known pyroglutamylated sulfopeptide QFPTDYDEGQDDRPK from the beta chain N-terminus was identified despite a complete lack of sulfate-containing fragment ions. The peptide QVGVEHHVEIEYD from the gamma-B chain C-terminus was also identified as sulfated or phosphorylated. This sulfopeptide is not annotated in Uniprot but was previously reported. MSFragger further identified a cysteine-containing peptide from the middle of the gamma chain as sulfated and deamidated. NanoLC-EThcD and LC-ECD MS/MS confirmed the two former sulfopeptides via sulfate-retaining fragment ions, whereas an unexpected fragmentation pattern was observed for the third sulfopeptide candidate. Manual interpretation of the LC-ECD spectrum revealed two additional isobaric identifications: a trisulfide-linked cysteinyl-glycine or a carbamidomethyl-dithiothreiotol covalent adduct. Synthesis of such adducts confirmed the latter identity.

Chemical Imaging in Vivo: Photoacoustic-Based 4-Dimensional Chemical Analysis.

We describe how 4-dimensional in vivo biochemical analysis can be performed using photoacoustic contrast nanoagents that have been designed to probe both structural and chemical information in vivo, enabling noninvasive, real time, spatially resolved chemical imaging. Early chemical imaging of a patient's tumor can inform the decision of effective treatment, regarding choices of chemotherapy, radiation, or immunotherapy.

Ion-Selective Nanosensor for Photoacoustic and Fluorescence Imaging of Potassium.

Ion-selective optodes (ISOs), the optical analog of ion-selective electrodes, have played an increasingly important role in chemical and biochemical analysis. Here we extend this technique to ion-selective photoacoustic optodes (ISPAOs) that serve at the same time as fluorescence-based ISOs, and apply it specifically to potassium (K+). Notably, the potassium ion is one of the most abundant cations in biological systems, involved in numerous physiological and pathological processes. Furthermore, it has been recently reported that the presence of abnormal extracellular potassium concentrations in tumors suppresses the immune responses and thus suppresses immunotherapy. However, unfortunately, sensors capable of providing potassium images in vivo are still a future proposition. Here, we prepared an ion-selective potassium nanosensor (NS) aimed at in vivo photoacoustic (PA) chemical imaging of the extracellular environment, while being also capable of fluorescence based intracellular ion-selective imaging. This potassium nanosensor (K+ NS) modulates its optical properties (absorbance and fluorescence) according to the potassium concentration. The K+ NS is capable of measuring potassium, in the range of 1 mM to 100 mM, with high sensitivity and selectivity, by ISPAO based measurements. Also, a near infrared dye surface modified K+ NS allows fluorescence-based potassium sensing in the range of 20 mM to 1 M. The K+ NS serves thus as both PA and fluorescence based nanosensor, with response across the biologically relevant K+ concentrations, from the extracellular 5 mM typical values (through PA imaging) to the intracellular 150 mM typical values (through fluorescence imaging).

Autofluorescence from NADH Conformations Associated with Different Metabolic Pathways Monitored Using Nanosecond-Gated Spectroscopy and Spectral Phasor Analysis.

Cellular NADH conformation is increasingly recognized as an endogenous optical biomarker and metabolic indicator. Recently, we reported a real-time approach for tracking metabolism on the basis of the quantification of UV-excited autofluorescence spectrum shape. Here, we use nanosecond-gated spectral acquisition, combined with spectrum-shape quantification, to monitor the long excited-state lifetime autofluorescence (usually associated with protein-bound NADH conformations) separately from the autofluorescence signal as a whole. We observe that the autofluorescence response induced by two NADH-oxidation inhibitors­cyanide and ethanol­are similar in Saccharomyces cerevisiae when monitored using time-integrated detection but easily distinguished using time-gated detection. Results are consistent with the observation of multiple NADH conformations as assessed using spectral phasor analysis. Further, because well-known oxidation inhibitors are used, changes in spectrum shape can be associated with NADH conformations involved in the different metabolic pathways, giving bioanalytic utility to the spectral responses.

Photoacoustic lifetime oxygen imaging of radiotherapy-induced tumor reoxygenation In Vivo.

Purpose: Early detection and diagnosis of cancer is critical for achieving positive therapeutic outcomes. Biomarkers that can provide clinicians with clues to the outcome of a given therapeutic course are highly desired. Oxygen is a small molecule that is nearly universally present in biological tissues and plays a critical role in the effectiveness of radiotherapies by reacting with DNA radicals and subsequently impairing cellular repair of double strand breaks.Techniques for measuring oxygen in biological tissues often use blood oxygen saturation to approximate the oxygen partial pressure in surrounding tissues despite the complex, nonlinear, and dynamic relationship between these two separate oxygen populations. Methods and materials: We combined a directly oxygen-sensitive, tumor-targeted, chemical contrast nanoelement with the photoacoustic lifetime-based (PALT) oxygen imaging technique to obtain image maps of oxygen in breast cancer tumors in vivo. The oxygen levels of patient-derived xenografts in a mouse model were characterized before and after a course of radiotherapy. Results: We show that, independent of tumor size, radiotherapy induced an increase in the overall oxygenation levels of the tumor. Further, this increase in the oxygenation of the tumor significantly correlated with a positive response to radiotherapy, as demonstrated by a reduction in tumor volume over the twenty-day monitoring period following therapy and histological staining. Conclusion: Our PALT imaging presented here is simple, fast, and non-invasive. Facilized by the PALT approach, imaging of tumor reoxygenation may be utilized as a simple, early indicator for evaluating cancer response to radiotherapy. Further characterization of the reoxygenation degree, temporal onset, and possible theragnostic implications are warranted.

Photoacoustic Chemical Imaging Sodium Nano-Sensor Utilizing a Solvatochromic Dye Transducer for In Vivo Application.

Sodium has many vital and diverse roles in the human body, including maintaining the cellular pH, generating action potential, and regulating osmotic pressure. In cancer, sodium dysregulation has been correlated with tumor growth, metastasis, and immune cell inhibition. However, most in vivo sodium measurements are performed via Na23 NMR, which is handicapped by slow acquisition times, a low spatial resolution (in mm), and low signal-to-noise ratios. We present here a plasticizer-free, ionophore-based sodium-sensing nanoparticle that utilizes a solvatochromic dye transducer to circumvent the pH cross-sensitivity of most previously reported sodium nano-sensors. We demonstrate that this nano-sensor is non-toxic, boasts a 200 µM detection limit, and is over 1000 times more selective for sodium than potassium. Further, the in vitro photoacoustic calibration curve presented demonstrates the potential of this nano-sensor for performing the in vivo chemical imaging of sodium over the entire physiologically relevant concentration range.

Photoacoustic Spectral Analysis for Evaluating the Aggressiveness of Prostate Cancer Labeled by Methylene Blue Polyacrylamide Nanoparticles.

Evaluating the aggressiveness of prostate cancer (PCa) is crucial for PCa diagnosis and prognosis. Previously, studies have shown that photoacoustic spectral analysis (PASA) can assess prostate tissue microarchitecture for evaluating the aggressiveness of PCa. In this study, in a transgenic mouse (TRAMP) model of PCa, we utilized methylene blue polyacrylamide nanoparticles (MB PAA NPs) to label the cancer cells in prostate in vivo. MB PAA NPs can specifically target proliferating cancer cells as a contrast agent, allowing photoacoustic (PA) imaging to better detect PCa tumors, and also assessing prostate glandular architecture. With the PA signals from the prostates measured simultaneously by a needle hydrophone and a PA and ultrasound (US) dual-imaging system, we conducted PASA and correlated the quantified spectral parameter slopes with the cancer grading from histopathology. The PASA results from 18 mice showed significant differences between normal and cancer, and also between low-score cancer and high-score cancer. This study in the clinically relevant TRAMP model of PCa demonstrated that PA imaging and PASA, powered by MB PAA NPs that can label the PCa microarchitectures in vivo after systemic administration, can detect PCa and, more importantly, evaluate cancer aggressiveness.

Personalized Oncology by In Vivo Chemical Imaging: Photoacoustic Mapping of Tumor Oxygen Predicts Radiotherapy Efficacy.

We hereby apply the approach of photoacoustic chemical imaging, performing an in vivo chemical analysis that is spatially resolved (200 µm) and in real time, to predict a given tumor's response to therapy. Using triple negative breast cancer as a model, we took photoacoustic images of tumors' oxygen distributions in patient-derived xenografts (PDXs) in mice using biocompatible, oxygen-sensitive tumor-targeted chemical contrast nanoelements (nanosonophores), which function as contrast agents for photoacoustic imaging. Following radiation therapy, we established a quantitatively significant correlation between the spatial distribution of the initial oxygen levels in the tumor and its spatial distribution of the therapy's efficacy: the lower the local oxygen, the lower the local radiation therapy efficacy. We thus provide a simple, noninvasive, and inexpensive method to both predict the efficacy of radiation therapy for a given tumor and identify treatment-resistant regions within the tumor's microenvironment.

Indocyanine green-enhanced multimodal photoacoustic microscopy and optical coherence tomography molecular imaging of choroidal neovascularization.

Photoacoustic microscopy (PAM) has great potential for visualization of the microvasculature with high spatial resolution and contrast. Early detection and differentiation of newly developed blood vessels named choroidal neovascularization (CNV) from normal vasculature remains a challenge in ophthalmology. Exogenous contrast agents can assist with improving PAM sensitivity, leading to differentiation of CNV. Here, an FDA-approved indocyanine green (ICG) was utilized as a PAM contrast agent. ICG was conjugated with RGD peptides, allowing the ICG to bind to the integrin expressed in CNV. Molecular PAM imaging showed that ICG-RGD can target CNV for up to 5 days post intravenous administration in living rabbits with a model of CNV. The PAM image sensitivity and image contrast were significantly enhanced by 15-fold at 24 h post-injection. Overall, the presented approach demonstrates the possibility of targeted ICG to be employed in PAM molecular imaging, allowing more precise evaluation of neovascularization.

Cell-morphodynamic phenotype classification with application to cancer metastasis using cell magnetorotation and machine-learning.

We define cell morphodynamics as the cell's time dependent morphology. It could be called the cell's shape shifting ability. To measure it we use a biomarker free, dynamic histology method, which is based on multiplexed Cell Magneto-Rotation and Machine Learning. We note that standard studies looking at cells immobilized on microscope slides cannot reveal their shape shifting, no more than pinned butterfly collections can reveal their flight patterns. Using cell magnetorotation, with the aid of cell embedded magnetic nanoparticles, our method allows each cell to move freely in 3 dimensions, with a rapid following of cell deformations in all 3-dimensions, so as to identify and classify a cell by its dynamic morphology. Using object recognition and machine learning algorithms, we continuously measure the real-time shape dynamics of each cell, where from we successfully resolve the inherent broad heterogeneity of the morphological phenotypes found in a given cancer cell population. In three illustrative experiments we have achieved clustering, differentiation, and identification of cells from (A) two distinct cell lines, (B) cells having gone through the epithelial-to-mesenchymal transition, and (C) cells differing only by their motility. This microfluidic method may enable a fast screening and identification of invasive cells, e.g., metastatic cancer cells, even in the absence of biomarkers, thus providing a rapid diagnostics and assessment protocol for effective personalized cancer therapy.

In vivo photoacoustic potassium imaging of the tumor microenvironment.

The accumulation of potassium (K+) in the tumor microenvironment (TME) has been recently shown to inhibit immune cell efficacy, and thus immunotherapy. Despite the abundance of K+ in the body, few ways exist to measure it in vivo. To address this technology gap, we combine an optical K+ nanosensor with photoacoustic (PA) imaging. Using multi-wavelength deconvolution, we are able to quantitatively evaluate the TME K+ concentration in vivo, and its distribution. Significantly elevated K+ levels were found in the TME, with an average concentration of approximately 29 mM, compared to 19 mM found in muscle. These PA measurements were confirmed by extraction of the tumor interstitial fluid and subsequent measurement via inductively coupled plasma mass spectrometry.

In Vivo Photoacoustic Lifetime Based Oxygen Imaging with Tumor Targeted G2 Polyacrylamide Nanosonophores.

Lifetime imaging methods using phosphorescence quenching by oxygen for molecular oxygen concentration measurement have been developed and used for noninvasive oxygen monitoring. This study reports photoacoustic (PA) oxygen imaging powered by polyacrylamide (PAAm) hydrogel nanoparticles (NP) which offer advantages including improved biocompatibility, reduced toxicity, and active tumor targeting. A known oxygen indicator, oxyphor G2, was conjugated with the matrix of the NPs, giving G2-PAA NPs, followed by PEGylation for biocompatibility and F3 surface modification for tumor targeting. Using two lasers providing pump and probe pulses, respectively, PA imaging was performed so as to quantitatively map the oxygen concentration in biological tissues in vivo, including cancer tumors and normal thigh muscles. Furthermore, via the imaging at the pump wavelength and two additional wavelengths, the accumulation of the G2-PAA NPs in the tumors were also determined. The successful imaging experiment accomplished on animal models renders a method for in vivo noninvasive imaging and assessment of hypoxic tumor microenvironments, which is critical for assessing cancer progression, metastasis, and treatment.