Up to date sample preparation of proteins for mass spectrometric analysis.

For the identification of proteins from biological samples by mass spectrometry there are two scenarios. Proteins are pre-fractionated in some way or are analysed as a complex mixture (shotgun). Shotgun proteomics recently became more popular, because of technological developments on the mass spectrometer side which now allows the identification of several thousand proteins from a complex biological matrix at one go. However, in many cases it is still useful to separate proteins first in a gel. But not only mass spectrometer technology made progress. Recently, new protocols and techniques which make the analysis of starting material in the low microgram range possible, and also simplify the whole procedure, were developed. In this small review detailed protocols will be described which also allow inexperienced beginners to get good results.


Introduction
Mass spectrometry became a valuable tool in biological analysis of proteins. During recent years the sensitivity of the instruments improved dramatically and this development is still ongoing. We are close being able to analyse whole proteomes of low microgram range in a single shot (shotgun analysis). The sample preparation becomes more and more important. It should be quick, relatively easy to perform, reproducible and with no, or minimal, loss of sample. Also recently protein sample preparation for mass spectrometry has made great progress towards the easy handling of small samples. Multistep procedures are usually not suited for starting material in the low microgram range. The loss of material during sample preparation is substantial. Therefore one step procedures are preferred. Inexperienced scientific staff are facing the problem of old protocols in the literature, incomplete handling procedures or protocols are scattered throughout several publications making it difficult to achieve a working protocol. The lack of important information or little ''tricks'' is the most common reason for irreproducible experiments. In this small review we will disclose all the details and following the protocols will produce good results instantly. We are using these protocols daily, but also in training courses for scientists and scientific staff.
In gel digestion (Eckerskorn & Lottspeich, 1989;Rosenfeld et al., 1992) of silver stained gels (Kerenyi & Gallyas, 1973) using the OASIS Õ HLB mElution Plate (Franz & Li, 2012) Protein separation in gels is still commonly used. For maximum sensitivity it is important to use a mass spectrometry compatible silver staining method (Chevallet et al., 2008). Manual de-staining of bands, reduction of cysteine bridges and alkylation of sulfides are labour intensive. Therefore we start with a manual high-throughput method for in gel digestion which will work for 1D gel bands as well as for 2D gel spots picked by a robot directly into the OASIS Õ HLB mElution Plate. The investment in the whole OASIS set up is only in the range of 5-10 chromatographic columns used for the mass spectrometric analysis.
The OASIS platform allows the use of an eight-channel pipette to add solutions, and with the help of the positive pressure stand (Waters, Milford, MA, USA) the solutions are then simultaneously removed. Thus, one can make a manual digest and de-salting of 96 samples in the same time and of the same quality that one could typically digest only 12 samples manually using standard 0.5 mL tubes. The samples were then de-stained, reduced, alkylated, digested, and the peptides extracted and desalted in the OASIS Õ HLB mElution Plate using the following protocol.

Methods
In-gel digestion by OASIS Õ HLB mElution Plate (Franz, 2012) (1) Use a spot cutter to excise the gel plugs from the 2D-gel directly into the OASIS Õ MTP well or alternatively cut a band by scalpel directly from the gel, place it at the upper rim of an OASIS Õ MTP well and cut it into three 1 mm pieces. Then push them down into the well by pipette tip. For 96 wells it will manually take about 1 h. (2) Add 70 mL acetonitrile (ACN) and push the gel pieces down into the solution if necessary. (3) Remove the liquid from the excision process into the waste plate (24 Â 10 mL MTP plate) using the positive pressure stand (PPS, if not otherwise stated 15 PSI of nitrogen pressure was always used). (4) Wait for 10 to 15 min until they have dehydrated (they will become small and white). If ACN runs through add a second round of 70 mL ACN after 5 min. (5) Using the PPS, remove all liquid into a waste plate.

Reduction
(1) Rehydrate gel pieces in 100 mL 10 mM DTT in 100 mM ammonium bicarbonate and incubate for 1 h at room temperature to reduce the cysteine bridges in the protein.
(2) Remove excess liquid using the PPS.
(3) Incubate the gel pieces with 100 mL ACN and wait for 10-15 min until the gel pieces have dehydrated, if ACN runs through add again. (4) Remove all liquid with the PPS.

Alkylation
(1) Swell gel pieces with 100 mL 55 mM IAA in 100 mM ammonium bicarbonate and incubate for 20 min at room temperature in the dark.
(2) Remove IAA solution with the PPS and wash gel pieces with 100 mL of 100 mM ammonium bicarbonate for 15 min. (3) Remove ammonium bicarbonate with the PPS, add 100 mL ACN and wait 10 min for the gel pieces to dehydrate and shrink, if ACN runs through add again after 5 min.

Application of trypsin
(1) Remove all liquid with the PPS, then rehydrate the gel pieces in 33 mL digestion buffer at room temperature.
(2) After 20 min remove remaining buffer with the PPS.
(3) Add 70 ml of the same buffer (50 mM ammonium bicarbonate), but prepared without trypsin, to cover the gel pieces and keep them wet during enzymatic digestion. (4) Leave samples covered at room temperature overnight or if available in an incubator at 37 C.

Extraction of peptides
(1) With the PPS remove the digest solution into the waste plate (peptides will bind to the HLB column). (2) Wash the OASIS Õ HLB column with 70 mL 0.1% FA.
(3) Exchange the waste plate for the collection plate (0.5 mL/well). (4) Add 50 mL of extraction solution (47.5% ACN/47.5% Water/5% FA) to Oasis plate and wait 20 min, then remove the extraction solution into collection plate with the PPS. (5) Add 50 mL of acetonitrile to OASIS plate and wait 10 min, then remove the extraction solution into collection plate with the PPS. (6) Completely dry eluted peptides in the collection plate using a vacuum centrifuge at 45 C for 60 min. (7) Freeze the dried extracts (peptides) at À20 C or À80 C (for storage 41 month) and/or (8) Re-dissolve the peptides in 7 mL 0.1% formic acid for ESI-MS or in 5 mL 0.1% TFA for MALDI-MS.
One step in solution protein solubilization, reduction, alkylation and in solution digest (Kulak et al., 2014) Shotgun approaches with very little starting material in the low microgram range can only be successful if sample loss is minimal and the solubilization of proteins is at the highest. Sample loss can be minimized by reducing the steps involved in the whole procedure. Usually for solubilizing proteins detergents are used, which are problematic in mass spectrometric analysis and involve a lot of steps to remove them.
Here we describe a very detailed protocol based on the latest Guanidinium method (Kulak et al., 2014) with a C. elegans sample as example.

Protocol:
It is important that the buffer is always fresh! First dissolve 5.73 g of GdmCl in 10 mL of Milli-Q water, then weight the other chemicals into a separate container and dissolve them completely with 3000 mL of the just prepared GdmCl solution and mix it with the rest of the GdmCl solution. As a rough estimation 100 mL of ''worm pellet'' corresponds to $ 1.8 mg peptide.
(2) Centrifuge the digest at 15 000 Â g for 10 min. Retain the supernatant. (3) For MS analysis the peptides have to be cleaned by STAGE (Rappsilber, 2003) or ZIP-Tip Õ (Millipore, Billerica, MA, USA). With this one-step sample processing we could identify around 4500 worm proteins with our standard 240 min gradient on a Q Exactive plusÔ instrument (Thermo).
If a maximum of proteins should be identified an easy to perform pre-fractionation routine can be done.
Pre-fractionation of protein digests with Styrene Divinyl Benzene (SDB-RPS) (Kulak et al., 2014) The SDB membrane containing tips are made in the same way as the STAGE tips (Rappsilber et al., 2003)  (1) The dried digest is re-suspended with 80 ml of 1% TFA in Milli Q Õ water. (2) Equilibrate the SDB tips with 200 mL of 0.2% TFA.
(3) Load 80 mL of the digest to the SDB tips and remove the liquid by centrifugation at 3000 Â g for 2 min. (4) Add 200 mL of 0.2% TFA for washing and centrifuge at 3000 Â g for 2 min. (5) Eluted the peptides with 80 mL of Buffer 1, Buffer 2, buffer 3 and Buffer 4 sequentially, and collect eluates separately in 0.5 mL Eppendorf tubes. With the combination of the four fractions (4 Â 150 min gradient duration) we were able to identify about 5600 worm proteins with a Q Exactive plusÔ and a 50 cm reversed phase column.

Conclusion
The technical development of mass spectrometers is very fast and also the sample preparation is continuously adapting to the needs of less starting material and more sensitivity. In this small review we show two good working protocols in every detail. With the detailed protocols beginners or advanced users will be quickly successful in the technically challenging analysis of complex proteomes.

Declaration of interest
The authors report no declarations of interest.