A major problem hampering the use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for quantitative measurements is the inhomogeneous distribution of analytes and matrices in solid sample preparations. The use of ionic liquids as matrices for the qualitative and quantitative analysis of low molecular weight compounds like amino acids, sugars and vitamins was investigated. The ionic liquid matrices are composed of equimolar combinations of classical MALDI matrices (sinapinic acid, alpha-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid) with organic bases. These matrix systems allow a homogenous sample preparation with a thin ionic liquid layer having negligible vapour pressure. This leads to a facilitated qualitative and quantitative measurement of the analytes compared with classical solid matrices.

The roles of four conserved basic amino acids in the reaction catalyzed by the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 have been investigated using site-directed mutagenesis in combination with measurements of steady-state kinetics, substrate-binding affinities and spectroscopic properties of the enzyme’s two prosthetic groups. Replacement of either Lys58 or Arg70 by glutamine leads to a complete loss of activity, with both the physiological electron donor, reduced ferredoxin and with a non-physiological electron donor, reduced methyl viologen. More conservative, charge-maintaining K58R and R70K variants were also completely inactive. Replacement of Lys130 by glutamine produced a variant that retained 26% of the wild-type activity with methyl viologen as the electron donor and 22% of the wild-type activity with ferredoxin as the electron donor, while replacement by arginine produces a variant that retains a significantly higher percentage of the wild-type activity with both electron donors. In contrast, replacement of Arg146 by glutamine had minimal effect on the activity of the enzyme. These results, along with substrate-binding and spectroscopic measurements, are discussed in terms of an in silico structural model for the enzyme.

Glycosylation is one of the most common post-translational modifications of proteins and plays essential roles in various biological processes, including protein folding, host-pathogen interaction, immune response, and inflammation and aberrant protein glycosylation is a well-known event in various disease states including cancer. As a result, it is critical to develop rapid and sensitive methods for the analysis of abnormal glycoproteins associated with diseases. Mass spectrometry in conjugation with different separation methods, such as capillary electrophoresis, ion mobility, and high performance liquid chromatography, has become a popular tool for glycoprotein analysis, providing highly informative fragments for structural identification of glycoproteins. This review provides an overview of the developments and accomplishments in the field of glycomics and glycoproteomics reported between 2014 and 2016.

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Ionic liquid matrixes (ILM) have been shown to allow very homogeneous sample preparations, facilitating relative quantifications using internal standards in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In the present work, the ability to perform quantifications of peptides without using internal standards in these matrixes was investigated. Linear correlations between peptide amount and signal intensities could be observed when increased molar matrix-to-analyte ratios were applied. The dynamic range of linearity was approximately 1 order of magnitude. The method was applied successfully to monitor the time-dependent evolution of substrates and products in trypsin-catalyzed digests of single peptides and peptide mixtures. Thus, ionic liquid matrixes allow quantitative MALDI-MS without the need for internal standards, making the method a suitable tool for the fast screening of new enzymes or the search for substrates or inhibitors.