Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry.

Sample preparation is the crucial starting point to obtain high-quality mass spectrometry data and can be divided into two main steps in a bottom-up proteomics approach: cell/tissue lysis with or without detergents and a(n) (in-solution) digest comprising denaturation, reduction, alkylation, and digesting of the proteins. Here, some important considerations, among others, are that the reagents used for sample preparation can inhibit the digestion enzyme (e.g., 0.1% sodium dodecyl sulfate [SDS] and 0.5 M guanidine HCl), give rise to ion suppression (e.g., polyethylene glycol [PEG]), be incompatible with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (e.g., SDS), and can induce additional modifications (e.g., urea). Taken together, all of these irreproducible effects are gradually becoming a problem when label-free quantitation of the samples is envisioned such as during the increasingly popular high-definition mass spectrometry (HDMS(E)) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) data-independent acquisition strategies. Here, we describe the detailed validation of a reproducible method with sufficient protein yield for sample preparation without any known LC-MS/MS interfering substances by using 1% sodium deoxycholate (SDC) during both cell lysis and in-solution digest.

Neutrophil Elastase in the capacity of the "H2A-specific protease".

The amino-terminal tail of histones and the carboxy-tail of histone H2A protrude from the nucleosome and can become modified by many different posttranslational modifications (PTM). During a mass spectrometric proteome analysis on haematopoietic cells we encountered a histone PTM that has received only little attention since its discovery over 35 years ago: truncation of the histone H2A C-tail at V114 which is mediated by the "H2A specific protease" (H2Asp). This enzyme is still referenced today but it was never identified. We first developed a sensitive AQUA approach for specific quantitation of the H2AV114 clipping. This clipping was found only in myeloid cells and further cellular fractionation lead to the annotation of the H2Asp as Neutrophil Elastase (NE). Ultimate proof was provided by NE incubation experiments and by studying histone extracts from NE Null mice. The annotation of the H2Asp not only is an indispensable first step in elucidating the potential biological role of this enzymatic interaction but equally provides the necessary background to critically revise earlier reports of H2A clipping.

Proteome characterization of human articular chondrocytes leads to novel insights in the function of small heat-shock proteins in chondrocyte homeostasis.

OBJECTIVE: In recent years, studies have been initiated to disclose the proteome of human chondrocytes and cartilage. Despite these studies, comprehensive information of the chondrocyte proteome remains limited. This study aimed to further explore the proteome expressed by human knee chondrocytes, and to study the functional aspects of heat-shock protein 27 (HSP27), a protein related to the previously described alphaBcrystallin, in chondrocyte biology. METHODS: Chondrocytes isolated from human knee articular cartilage were cultured in a three-dimensional alginate culture system. To simplify the protein mixtures, proteins extracted from chondrocyte cell lysates were fractionated based on hydrophobicity and molecular weight. Proteins were digested and the resulting peptides were separated and identified by an on-line two-dimensional (2-D) nanoliquid chromatography (nanoLC)-system coupled to a quadrupole time-of-flight (Qq-TOF) mass spectrometer. Differential expression analysis of HSP27 was performed by Western Blotting and quantitative polymerase chain reaction (QPCR). The effects of HSP27 on chondrocyte biology were explored by suppression of HSP27 expression induced by RNA interference (RNAi). RESULTS: In this study, we identified proteins with unknown functions together with membrane proteins, transcription factors and other low abundant proteins, which have not yet been described in chondrocytes. Based on previous knowledge on the related protein alphaBcrystallin, we selected HSP27 from the chondrocyte proteome database. Differential expression analysis revealed a decreased expression of HSP27 in Osteoarthritic (OA) chondrocytes. RNAi experiments revealed that HSP27 is involved in interleukin-1beta (IL-1beta) induced IL-6 secretion. CONCLUSION: These findings highlight that small HSPs, especially HSP27, play a prominent role in the maintenance of human articular chondrocyte homeostasis.