Ultrasonic Enhanced Applications in Proteomics Workflows: single probe versus multiprobe.

Journal of Integrated Omics *Corresponding author: Jose. L. Capelo. Tel.: +34 610 835 903; Email Address: jlcapelom@uvigo.es; URL: http://www.bioscopegroup.com (J.L. Capelo). | DOI: 10.5584/jiomics.v1i1.55 Luz Fernandez et al., 2010 | Journal of Integrated Omics 144-150: 145 jet II was used for (i) sample drying and (ii) sample preconcentration. A minicentrifuge, model Spectrafuge-mini, from Labnet (Madrid, Spain), and a minicentrifuge-vortex, model Sky Line, from ELMI (Riga, Latvia) were used throughout the sample treatment, when necessary. A Simplicity 185 from Millipore (Milan, Italy) was used to obtain Milli-Q water throughout the experiments. 2.2 Ultrasonic devices (i) Ultrasonic probe, model UP 100H (dr. Hielscher, Teltow, Switzerland, 200 W, 30 kHz ultrasonic frequency, 0.5 mm of diameter probe). (ii) Ultrasonic multi-probe from Branson Ultrasonics Corporation (USA), model SLPe (150 W, 40 kHz ultrasonic frequency, 1 mm diameter probe). The ultrasonic generator SLPe is equipped with a multi-probe detachable horn (model 4c15), with four tips for simultaneous ultrasonication of four samples and it was used in conjunction with a 96-well plate, as it is depicted in video 1 of supporting information. 2.3 Standards and reagents The following protein standards were used: α-lactalbumin from bovine milk (≥85%), BSA (>97%) and carbonic anhydrase (>93%) from Sigma (Steinheim, Germany), albumin from hen white (>95%) from Fluka (Buchs, Switzerland). Chymotrypsinogen A, catalase bovine and aldolase from rabbit were standards for gel filtration calibration kit high molecular weight from Amersham Biosciences (Piscataway, USA). Low molecular weight standard protein mixture of glycogen phosphorylase b, bovine serum albumin, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor and α-lactalbumin were purchased from Amersham Biosciences (Piscataway, USA). Thyroglobulin and Lactate dehydrogenase were purchased from Amersham Biosciences (Piscataway, USA) Carp vitellogenin standard was purchased from Biosense Laboratories (Bergen, Norway). Trypsin enzyme, sequencing grade was purchased from Sigma. All materials were used without further purification. α-cyano-4-hydroxycinnamic acid (α-CHCA) puriss for MALDI–MS from Fluka was used as MALDI matrix. ProteoMassTM Peptide MALDI-MS Calibration Kit (MSCAL2) from Sigma was used as mass calibration standard for MALDITOF-MS. The following reagents were used for protein depletion: sodium chloride puriss. p.a. and magnesium chloride hexahydrate puriss. p.a. were purchased from Fluka; ethylenediaminetetraacetic acid disodium salt dehydrate puriss. p.a. was from Riedle-de Haen (Seelze, Germany). The following reagents were used for protein digestion: acetonitrile, iodoacetamide (IAA) and DL-dithiothreitol (DTT) (99%) were purchased from Sigma; formic acid and ammonium bicarbonate (>99.5%) were from Fluka; trifluoroacetic acid (TFA, 99%) were from Riedel-de-Haen (Seelze, Germany); and urea (99%) was from Panreac (Barcelona, Spain). 2.4 Sample treatment 2.4.1. Protein separation by 1D-SDS-PAGE Amounts of protein ranging from 0.5 to 3.7 μg were dissolved in 5 μL of water plus 5 μL of sample buffer (5 mL of 0.5 M Tris-Base + 8 mL of 10 % SDS + 1 mL of βmercaptoethanol + 2 mL of glycerol + 4 mg of bromophenol blue in a final volume of 20 mL in water) for analysis by sodium dodecyl sulphate polyacrilamide gel electrophoresis (SDS-PAGE) (10% 0.5 mm thickness). After gel electrophoresis (65 min, 120 V, 400 mA), the gel was stained with Coomassie blue R-250 and destained in order to visualize the proteins bands. 2.4.2. In-gel sample treatments (i) Overnight method. For in-gel digestion optimization, 2.1 μg of BSA and 2.9 μg of α-lactalbumin were loaded onto 10% SDS-PAGE gels. Coomassie Blue-stained protein bands were excised from the gels, cut into pieces and subjected to digestion. Excised gel bands were then washed with water (3 times with agitation/centrifugation, 10min each), and dehydrated with acetonitrile (2 times, 3 min each + 1 time, 20 min with agitation/centrifugation) and dried in a vacuum centrifuge. Gel pieces were further rehydrated with 10 mM of DTT in 25 mM ammonium bicarbonate buffer and incubated 10 min at 60 oC for protein reduction. Then DTT solution was replaced by IAA 55 mM in 25 mM ammonium bicarbonate buffer and incubated in the dark and room temperature by 35 min. After protein reduction and alkylation gel pieces were dried and rehydrated in ice bath in a 0.025 μg/μL solution of trypsin in 12.5 mM ammonium bicarbonate buffer, to a final volume of 25 μL, during 1 h. After the rehydration step, samples were digested overnight at 37 °C. Next, trypsin activity was stopped by the addition of 20 μL of 5% formic acid. The supernatant was withdrawn and retained, and the peptides were extracted from the gel pieces by adding 50-100 μL of a mixture of acetonitrile/TFA (500 μL H2O+500 μL AC+1 μL TFA) and incubating them for 15 min at 37 oC in a shaker. This extraction was done twice. Then, all supernatants were combined and evaporated to dryness in a vacuum concentrator centrifuge and finally the dried peptides obtained were reconstituted with 10 μL of 0.3% v/v formic acid. (ii) Accelerated method. In this method, the protocol described above and referred as “overnight method” was followed but (i) washing steps (ii) alkylation, (iii) reduction, (iv) digestion of gel bands and (v) extraction peptides were done in 8 min (2 min each washing step, 30% ultrasonic amplitude, total of 3), 5 min (30% ultrasonic amplitude), 5 min (30% ultrasonic amplitude), 4 min (25% ultrasonic amplitude) and 8 min (two extraction steps, 2 min each) respectively, using ultrasonication at 30 kHz, with a single probe or 40 kHz with the four tip multiprobe.