ZipTip Sampling

To overcome problems with automation and recovery, ZipTip sampling has been developed, which is best regarded as miniaturized and simplified SPE equipment. It is frequently used in the field of proteomics, mostly as the last sample preparation step. Sample amount is only a few (3-30) p,l, containing less than a picomole protein digest. Commonly it is used to remove salts and detergents from the sample, e.g., after tryptic digest, just before mass spectro-metric analysis. 

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The quality of the sample influences the quality of the data obtained by MALDI-TOFF. Contaminations inhibit ionization. As no in-line HPLC system is included in the system, contaminants cannot be removed from the sample. Fortunately, some new clean up tools (e.g., ZipTips) for removal of contaminants have been developed recently (Figure 5.5). 

A versatile approach to nano-ESl is the use of a microchip containing an array of 10x10 nano-ESl emitter tips, typically 8 pm ID. A sample-handling robot performs continuous infusion of samples from a 96-well or 384-well plate using a new emitter tip at the chip for each individual sample. This system is commercially available from Advion BioSciences and has found wide application. An example is the identification of proteins excised from a 2D gel electrophoresis gel and in-gel digested prior to infusion [7]. Implementation of ZipTip -based sample pretreatment strategies in combination with these nano-ESl microchips was reported as well [8]. 

Dry samples using a SpeedVac, dissolve in a small volume (3 pi) of saturated 4HCCA matrix solution, and analyze by MALDI-TOF MS (see Section 2.6.3). Alternatively, desalt samples using RP C18 ZipTips and analyze by ESI MS. 

Desalt sulfotyrosine peptide samples by binding to RP-C18 ZipTips according to the manufacturer s protocol. After washing with HPLC-grade water, elute peptides with 70% acetonitrile. 

After equilibration, bind peptides to ZipTip by fully depressing the pipettor plunger to a dead stop. Aspirate and dispense sample 3-7 cycles for simple mixtures and up to 10 cycles for maximum binding of complex mixtures. 

Optimal binding of protein to ZipTip may also require a chao-tropic agent (e.g., urea or guanidine-HCl at a final concentration of approximately M). If sample does not already contain a chaotropic salt, add a few drops of salt before binding. In the case of excess detergent, dilute sample with 0.1% TFA to achieve acceptable binding conditions, e.g., SDS (<0.1 %), Triton (<1 %), and Tween (<0.5 %). 

Finally, the transcripts were completely digested by RNase Ti at 37°C for 10 min with MALDI matrix (3-HPA) added as a denaturant. Briefly, 5 pL of RNA transcript (up to 20 pg) is added to 4 pL 3-HPA in 50% ACN/water and 1 pL of RNase Tj (1000 units) and reacted for 10 min and then placed on ice for MALDI preparation. For highest quality spectra, samples are desalted by reverse-phase purification in ZipTips according to manufacturer s directions for nucleic acid purification [96]. The final step in this process is elution of purified RNA oligonucleotide fragments onto the MALDI target using 2 pL of the MALDI matrix itself. Samples (MALDI spots ) are allowed to air dry or may be rapidly dried under vacuum. 

Another approach is to use tips of microliter pipettes as miniature columns by filling them with size exclusion chromatography (SEC) stationary phase material or standard Cig reversed phase material [125,126]. Such tips are also commercially available as ZipTips [122]. There is a risk of sample absorption on the... 

Ultrapure water (18.2 MS2 cm), was obtained from a MiUipore water purification system and was used for preparation of sample solutions. Analytical Grade Acetonitrile (MeCN) (Fischer Scientific) and Trifluoroacetic Acid (TEA) (Sigma-Aldrich) were used for all ZipTip buffers (see Sect. Denaturing MS— ZipTip Buffers ). The coenzyme A derivatives (acetyl-, butyryl- and malonyl) used in assays were purchased from Sigma-Aldrich. iV-acetylcysteamine (SNAC) thioesters were provided by the Piel lab (ETH, Zurich), and stock solutions were solubilised in dimethyl sulfoxide (DMSO) (Fischer Scientific). lodoacetamide (Sigma-Aldrich) was used for all alkylation reactions. 

KS loading assays were conducted in storage buffer (25 mM Tris, 500 mM NaCl 10 % (v/v) glycerol, pH 7.6). Typically, 20 p,M KS was incubated with 100 ttM acyl-ACP and the reaction was allowed to proceed at 25 °C. Aliquots were removed at various time points between 2-32 h, and subsequent ZipTip clean-up of the samples allowed MS analysis of each time point (see Sect. 2.2.15.1). 

Both Ci8 and C4 ZipTips i were used to prepare samples the Cis was primarily used for desalting of the ACP, whereas C4 was used for KS and AT domains. The ZipTip was washed with two 10 xL aspirations of 50 % MeCN, followed by five 10 tiL aspirations of H2O/O.I % TFA solution. The protein sample was then loaded onto the ZipTip column by 15-20x 10 tiL aspirations of the sample, each time injecting back into the sample vial. The loaded protein sample was then desalted by xl5 10 xL aspirations of 0.1 % TFA, followed by elution of the sample into 8 xL H2O/8O % MeCN/0.1 % TFA. 

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