Several proteomics approaches are available that are defined by the level (protein or peptide) at which analysis takes place. The most widely applied method still is bottom-up proteomics where the protein is digested into peptides that can be efficiently analyzed with a wide range of LC-MS or MALDI-TOF-MS instruments. Sample preparation for bottom-up proteomics experiments requires several treatment steps in order to get from the protein to the peptide level and can be very laborious. The most crucial step in such approaches is the protein digestion, which is often the bottleneck in terms of time consumption. Therefore, a significant gain in throughput may be obtained by speeding up the digestion process. Current techniques allow for reduction of the digestion time from overnight (~15 h) to minutes or even seconds. This advancement also makes integration into online systems feasible, thereby reducing the number of tedious sample handling steps and the risk of sample loss. In this review, an overview is given of the currently available digestion strategies and recent developments in the acceleration of the digestion process. Additionally, tailored approaches for classes of proteins that pose specific challenges are discussed.

An accurate mass measurement of a known protein provides information on potential amino acid deletions and post-translational modifications. Although this field is dominated by strategies based on electrospray ionization, mass spectrometry (MS) methods using matrix-assisted laser desorption/ionization (MALDI) have the advantage of yielding predominantly singly charged precursor ions, thus avoiding peak overlap from different charge states of multiple species. Such MALDI-MS methods require mass measurement at ultrahigh resolution, which is provided by Fourier transform ion cyclotron resonance (FTICR) mass analyzers. Recently, using a MALDI-FTICR-MS platform equipped with a 15 T magnet, we reported on the mass analysis of intact human serum peptides and small proteins with isotopic resolution up to ∼15 kDa and identified new proteoforms from an accurate measurement of mass distances. In the current study, we have used this FTICR system after an upgrade with a novel dynamically harmonized ICR cell, i.e., ParaCell, for mapping isotopically resolved intact proteins up to about 17 kDa and performed top-down MALDI in-source decay (ISD) analysis. Standard proteins myoglobin (m/z-value 16,950) and ribonuclease B (m/z-value 14,900) were measured with resolving powers of 62,000 and 61,000, respectively. Furthermore, it will be shown that (singly charged) MALDI-ISD fragment ions can be measured at isotopic resolution up to m/z-value 12,000 (e.g., resolving power 39,000 at m/z-value 12,000) providing more reliable identifications. Moreover, examples are presented of pseudo-MS(3) experiments on ISD fragment ions from RNase B by collisional-induced dissociation (CID).

A high-throughput sample preparation protocol based on the use of 96-well molecular weight cutoff (MWCO) filter plates was developed for shotgun proteomics of cell lysates. All sample preparation steps, including cell lysis, buffer exchange, protein denaturation, reduction, alkylation and proteolytic digestion are performed in a 96-well plate format, making the platform extremely well suited for processing large numbers of samples and directly compatible with functional assays for cellular proteomics. In addition, the usage of a single plate for all sample preparation steps following cell lysis reduces potential samples losses and allows for automation. The MWCO filter also enables sample concentration, thereby increasing the overall sensitivity, and implementation of washing steps involving organic solvents, for example, to remove cell membranes constituents. The optimized protocol allowed for higher throughput with improved sensitivity in terms of the number of identified cellular proteins when compared to an established protocol employing gel-filtration columns.

Liquid chromatography−mass spectrometry (LC−MS) has been widely used throughout biotherapeutic development. However, its implementation in GMP-compliant commercial quality control (QC) laboratories remains a challenge. In this publication, we describe the covalidation and implementation of an automated, high-throughput, and GMP compliant subunit LC−MS method for monitoring antibody oxidation for commercial product release and stability testing. To our knowledge, this is the first report describing the implementation of a high-resolution LC−MS method in commercial QC laboratories for product release and stability testing in the biopharmaceutical industry. This work paves the road for implementing additional LC−MS methods to modernize testing in commercial QC with more targeted control of product quality.

Disulfide bonds are an important class of protein post-translational modifications, yet this structurally crucial modification type is commonly overlooked in mass spectrometry (MS)-based proteomics approaches. Recently, the benefits of online electrochemistry-assisted reduction of protein S–S bonds prior to MS analysis were exemplified by successful characterization of disulfide bonds in peptides and small proteins. In the current study, we have combined liquid chromatography (LC) with electrochemistry (EC) and mass analysis by Fourier transform ion cyclotron resonance (FTICR) MS in an online LC-EC-MS platform to characterize protein disulfide bonds in a bottom-up proteomics workflow. A key advantage of a LC-based strategy is the use of the retention time in identifying both intra- and interpeptide disulfide bonds. This is demonstrated by performing two sequential analyses of a certain protein digest, once without and once with electrochemical reduction. In this way, the “parent” disulfide-linked peptide detected in the first run has a retention time-based correlation with the EC-reduced peptides detected in the second run, thus simplifying disulfide bond mapping. Using this platform, both inter- and intra-disulfide-linked peptides were characterized in two different proteins, ß-lactoglobulin and ribonuclease B. In order to prevent disulfide reshuffling during the digestion process, proteins were digested at a relatively low pH, using (a combination of) the high specificity proteases trypsin and Glu-C. With this approach, disulfide bonds in ß-lactoglobulin and ribonuclease B were comprehensively identified and localized, showing that online LC-EC-MS is a useful tool for the characterization of protein disulfide bonds.Graphical AbstractᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s13361-015-1258-z) contains supplementary material, which is available to authorized users.