On-plate digestion using a commercial microfraction collector for nano-HPLC matrix-assisted laser desorption/ionization tandem time-of-flight protein analysis

Abstract

A new method for on-plate protein digestion and MALDI MS analysis is proposed involving an automated one-step sample separation using nanoflow-HPLC followed by nanoliter fraction collection and on-plate digestion with trypsin. This procedure uses a commercial automatic nanoliter fraction collection system for on-line spotting of the eluent onto a MALDI target. After protein digestion, the reaction is stopped by the addition of acidified matrix using the same automated system. Collected spots are subsequently analyzed using a MALDI-TOF/TOF mass spectrometer for protein sequencing and identification. Keywordsacquired enamel pellicle; LC-MS/MS; on-plate digestion; proteomic; MALDI-TOF/TOF Gel-free methods have been developed based on liquid separations coupled on-line with mass spectrometry (LC-MS) for proteomic analyses of complex biological systems (1);(2)). The sequence information obtained from peptide fragment fingerprints and database searching is often sufficient for identification (1-3). However, one of the limitations of this approach is the difficulty in identification of protein isoforms or highly homologous proteins. One approach to circumvent this problem proposed by Zheng et al. (4), is based on protein separation using a capillary column followed by fraction collection on MALDI targets pre-coated with trypsin for on-plate digestion, followed by MALDI-TOF/TOF analysis.We have developed a new "hands-off" method for on-line protein digestion and MALDI analysis based on a robotic system coupled to a nano-HPLC. This system offers improved reproducibility and ease of handling for routine proteomics studies using standard gel-free mass spectrometry and tandem mass spectrometry screening procedures. In order to assess this approach, we performed on-plate plate digestion with standard proteins such as equine myoglobin (Sigma M0630), bovine insulin (Sigma, St. Louis, MO, I5500) and bovine serum albumin (Sigma L3908). Protein solutions were prepared at an initial concentration of 1mg/ mL and then diluted, so that 0.5 μL of the protein solution corresponded to 20 pmol, 100 pmol, 200 pmol, 600 pmol and 12 nmol of protein. Aliquots, 0.5 μL were spotted onto a MALDI target, and the spot was then incubated with 0.2 μL of a trypsin solution (0.2 μg/μL in 25mMCorrespondence, Rui M P Vitorino, Department of Chemistry, University of Aveiro, Aveiro, Portugal, rvitorino@ua. The database search parameters were: mass tolerance of 40 ppm for precursor ions and 0.3 Da for fragment ions; trypsin digestion with two missed cleavages; variable modifications such as N -terminal Gln, pyroGlutamic acid, methionine oxidation, N-terminal acetylation and phosphorylation of serine, threonine and tyrosine residues. A positive identification was accepted at the 95% confidence level.As expected, good protein coverage was achieved through the approach of in-solution digestion of the protein standards at all tested concentrations. Similar results were obtained using our protein on-plate digestion approach based on the number of detec...

On‐plate deposition of oxidized proteins to facilitate protein footprinting studies by radical probe mass spectrometry

“…After protein deposition on to the grounded MALDI plate, the protein was subjected to on‐plate digestion. This involved redissolving the protein in ammonium bicarbonate buffer prior to performing successive on‐plate cysteine reduction and proteolytic digestion . The MALDI mass spectrum of the tryptic digest of lysozyme oxidized at 6 kV is shown in Fig.…”

“…To speed the analysis of proteins oxidized by RP‐MS, and avoid the need to collect and reconstitute proteins prior to analysis, we describe here coupling the electrical discharge oxidation of proteins with on‐plate deposition for matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). On‐plate sample collection for MALDI has been combined with liquid chromatography (LC) and capillary electrophoresis (CE), while on‐plate digestion of proteins has also been achieved . Furthermore, on‐plate chemical oxidation has been reported as a means to identify methionine containing peptides .…”

“…On-plate sample collection for MALDI has been combined with liquid chromatography (LC) [16] and capillary electrophoresis (CE), [17] while on-plate digestion of proteins has also been achieved. [18,19] Furthermore, on-plate chemical oxidation has been reported as a means to identify methionine containing peptides. [20] The ability to deposit oxidized protein directly onto MALDI sample plates improves sample throughput enabling more protein samples to be oxidized and analyzed in succession.…”

Ratiometric Raman spectroscopy for quantification of protein oxidative damage

“…Since DCDR is a nondestructive technique, the same protein deposit on the stainless steel plate can be directly used for mass spectrometric analysis after DCDR is completed[23]. On plate trypsin digestion, followed by MALDI-TOF, protein identification has been demonstrated by Vitorino et al [39]. …”

Lab‐on‐a‐plate: Extending the functionality of MALDI‐MS and LDI‐MS targets

“…Automation of the protocol for on-plate digestion with trypsin was recently proposed using a commercial fraction collector for high performance liquid chromatography (HPLC) (Getie-Kebtie et al, 2008) and nanoflow HPLC (Vitorino et al, 2008). Importantly, a significant improvement of sensitivity and robustness of this protocol was demonstrated.…”