Nanocapillary liquid chromatography interfaced to tandem matrix‐assisted laser desorption/ionization and electrospray ionization‐mass spectrometry: Mapping the nuclear proteome of human fibroblasts

Abstract

Miniaturized liquid chromatography nanoseparation in combination with minigel fractionation of human primary cell nuclei is presented. We obtained high-sensitivity and high-throughput identification of expressed proteins by subcellular fractionation and nanocapillary liquid chromatography interfaced to both electrospray ionization (ESI)- and matrix-assisted laser desorption/ionisation (MALDI) tandem mass spectrometry. The reversed-phase nanocapillary eluents were applied directly onto the MALDI target plate as discrete crystal spots using in-line matrix infusion. When working with primary cells, only a limited amount of sample is available. To maximize the number of identified proteins from a restricted amount of sample, miniaturized sample preparation protocols and nanoflow separation is a necessity, especially when working with low-abundant proteins. From the same isolated nuclear sample, complementary separation of intact proteins by two-dimensional (2-D) gel electrophoresis was made. In total 594 gene products from the nuclear preparations were identified out of which 261 were unique. Several proteins involved in transcriptional events were identified such as TATA-binding protein, EBNA-co-activator, and interleukin enhancer binding proteins, indicating that sufficient proteomic depth is obtained to study transcriptional controlling events. Our results suggest that by sample prefractionation and downscaled nanoflow separation along with a combined mass spectrometry strategy, it is possible to identify a large number of nuclear proteins from human primary cells. These findings are of particular importance due to the disease link of these targets cells.

New directions of miniaturization within the proteomics research area

“…These investigations were made in human cells, where thousands of proteins were identified. We could conclude that MALDI-TOF/TOF was superior in comparison with ESI where we reached an identity (ID)-rate of about 75% of the total number of annotations made in the study [17,18].…”

“…On the contrary, these modes of sequencing are complementary to each other. We have found in proteomics studies [17,18] that while the unique expression and identification is a fact, there is also an identity overlap where the two ionization principles make successful annotation. Capillary liquid chromatography (Cap-LC) is favorable for tandem MS where the on-line configuration of real-time sequencing is a big advantage.…”

“…Two-dimensional nano-LC/MS systems were also developed [24] being able to identify hundreds of proteins significantly regulated in a yeast study. An alternative to multidimensional separation is a combination of 1-D gels as first separation step, followed by in-gel digestion of the protein bands from this first step and reversed-phase nanocapillary-LC separation [17,25,26 ] (Fig. 3).…”

“…The success rate of protein identification at the moment with the TOF/TOF reaches an efficiency of over 90% [31]. TOF/TOF was first introduced with a quadrupole linear configuration [32] showing the ability to sequence peptides in human primary cells where the identity was not possible by MALDI-PMF [17]. It is also clear that one single ionization principle is unable to identify a large set of proteins while the combination of both ESI and MALDI can.…”

“…The subcellular isolation of the cell nuclei for which the methodology for high-density isolations has been developed, was found to be critical in order to generate quantitative expression analysis studies in search of lower-abundant protein regulation. Figure 5A presents the primary human cells used in these studies where the growth factor activation has a profound impact on the phenotype [17,18]. The cell morphology in this case will change completely.…”