Cholesterol transport between cellular organelles comprised vesicular trafficking and nonvesicular exchange; these processes are often studied by quantitative fluorescence microscopy. A major challenge for using this approach is producing analogs of cholesterol with suitable brightness and structural and chemical properties comparable with those of cholesterol. This review surveys currently used fluorescent sterols with respect to their behavior in model membranes, their photophysical properties, as well as their transport and metabolism in cells. In the first part, several intrinsically fluorescent sterols, such as dehydroergosterol or cholestatrienol, are discussed. These polyene sterols (P-sterols) contain three conjugated double bonds in the steroid ring system, giving them slight fluorescence in ultraviolet light. We discuss the properties of P-sterols relative to cholesterol, outline their chemical synthesis, and explain how to image them in living cells and organisms. In particular, we show that P-sterol esters inserted into low-density lipoprotein can be tracked in the fibroblasts of Niemann–Pick disease using high-resolution deconvolution microscopy. We also describe fluorophore-tagged cholesterol probes, such as BODIPY-, NBD-, Dansyl-, or Pyrene-tagged cholesterol, and eventual esters of these analogs. Finally, we survey the latest developments in the synthesis and use of alkyne cholesterol analogs to be labeled with fluorophores by click chemistry and discuss the potential of all approaches for future applications.

Exploring post-translational modifications (PTMs) with the use of mass spectrometry (PTMomics) is a rapidly developing area, with methods for discovery/quantification being developed and advanced on a regular basis. PTMs are highly important for the regulation of protein function, interaction and activity, both in physiological and disease states. Changes in PTMs can either cause, or be the result of a disease, making them central for biomarker studies and studies of disease pathogenesis. Recently, it became possible to study multiple PTMs simultaneously from low amount of sample material, thereby increasing coverage of the PTMome obtainable from a single sample. Thus, quantitative PTMomics holds great potential to discover biomarkers from tissue and body fluids as well as elucidating disease mechanisms through characterization of signaling pathways. Areas covered: Recent mass spectrometry-based methods for assessment of the PTMome, with focus on the most studied PTMs, are highlighted. Furthermore, both data dependent and data independent acquisition methods are evaluated. Finally, current challenges in the field are discussed. Expert commentary: PTMomics holds great potential for clinical and biomedical research, especially with the generation of spectral libraries of peptides and PTMs from individual patients (permanent PTM maps) for use in personalized medicine.

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Type 2 diabetes mellitus (T2DM) is a complex group of disorders, characterized by hyperglycemia, insulin resistance and insulin deficiency. In human blood, hyperglycemia ultimately results in the enhancement of glycation -a posttranslational modification formed by the interaction of protein amino groups with glucose. The resulting fructosamines (Amadori compounds) readily undergo further degradation resulting in advanced glycation end products (AGEs), known to be pro-inflammatory in humans. These compounds are highly heterogeneous and characteristic of advanced stages of the disease, whereas fructosamines are recognized markers of early diabetes stages (HbA 1C , glycated albumin). Recently, individual plasma protein glycation sites were proposed as promising T2DM biomarkers sensitive to short-term fluctuations of plasma glucose. However, corresponding absolute quantification strategies, applicable in regular clinical practice, are still not established. Therefore, here we propose a new analytical approach aiming at reproducible and precise quantification of multiple glycated peptides in human plasma tryptic digests. Thereby, the standard peptides comprised a 13 C, 15 N-labeled lysyl residue, a dabsyl moiety for determination of standard amounts, and a cleavable linker. Known amounts of these peptides were spiked to plasma samples prior to tryptic digestion, quantification relying on stable isotope dilution. The method was demonstrated to be applicable for quantification of individual glycated sites in T2DM patients and non-diabetic controls.

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