Analysis of proteins in complex mixtures on a Ziptip via mass spectrometry provides a rapid, quantitative means to assay noncovalent modifications directly from tissues that is comparable and possibly more sensitive than western blotting. The remarkable sensitivity of the method allows for anatomical microdissection of the tissue. ALS affects motor neurons, which are localized in the ventral spinal cord (). This assay allows SOD1 to be directly measured from the disease-affected regions, and is able to distinguish between one-, two-, and no-metal bound states; the one-metal bound species is significantly larger in the ventral compared to the dorsal spinal cord.This difference is lost if the entire spinal cord is used, as several previous methods have done[33
Three main issues needed to be addressed in developing the assay. The first was to maintain metal binding to the protein while providing sufficient acidity to permit efficient electrospray ionization [36
]. We found that 0.1% formic acid, commonly used in ESI-MS coupled liquid chromatography, was too strong and could cause SOD1 to lose metals during the assay [32
]. Hayward et al.
, used 0.1% formic acid to look at apo mutant SOD1 proteins [25
].Leaving out formic acid resulted in poor ionization because the solvent could not provide enough hydrogen ions in solution to effectively ionize SOD1. The largest amounts of human Cu,Zn SOD1 eluting from the Ziptip were in the range of 1-10 pmol in a volume of a few microliters, resulting in a concentration of 1-10 μM. Because the charge states of SOD1 typically ranged from 8 to 13, the concentration of hydrogen ions needed to be 8-130 μM (). We found that solvent containing 100 μM formic aciddelivered at a flow rate of 30 μL/minprovided enough protons for efficient ionization. The 100 μM formic acid concentration is approximately 200-fold more dilute than the standard 0.1% concentration, and was able to efficiently ionize SOD1 without altering metal binding after five minutes, twice the time needed to run the assay ().
The second issue to overcome was how to efficiently remove salts from the protein solution that would otherwise interfere with electrospray ionization. Salts suppress ionization of proteins, interfering with the deconvolution of raw m/z data (). Sodium ions in particular can associate strongly with an ionizing protein and sodium chloride is approximately 100 mM in the central nervous system, thereby presenting a significant potential interference to ionizing proteins directly from tissue. We used a C4Ziptipto desalt the samples. The Ziptip is a P10 pipette tip with a small amount of HPLC reversed-phase packing material. As tissue lysate is drawn over it, soluble proteins bind to the packing material. Once bound, the proteins could be rinsed multiple times with water, washing off salts and other small hydrophilic molecules while leaving the proteins bound to the packing material. The bound proteins could be eluted in the mass spectrometer with the minimal concentration of acetonitrile needed to elute SOD1 (30%) ().
The third issue was how to quantify the amount of human SOD1 released from the tissue. This was accomplished by including an internal standard, bovine SOD1, at a known concentration in the punch thawing buffer. Comparing the integration of the peak areas from MOP™ of the bovine and human SOD1 allowed us to calculate the amount of human SOD1 in the punch supernatant, which could be used to estimate the concentration of human SOD1 in the tissue.SOD1 was found to be 16pmol/mg tissue in the dorsal spinal cord and 26pmol/mg tissue in the ventral spinal cord. These amounts roughly translate to 16 and 26μM in the cells, respectively. A standard curve of recombinant human SOD1 compared to bovine SOD1 was used to determine if the ionization of the human and bovine proteins differed (). The slope of 1.3 on the standard curve suggests that human SOD1 may ionize more efficiently than bovine SOD1 due to the higher number of basic amino acid residues present in the human SOD1 sequence. This can also be seen in the differing charge state distributions of human and bovine SOD1, in which the human protein picks up two more positive charges on average than bovine SOD1 ().
One of the primary advantages of our method is the ability to monitor the metal-binding of SOD1 at the monomer level in complex mixtures, in contrast to other approaches that can determine the metal-binding of the population of SOD1 as a whole solution, thus giving only average stoichiometry [35
]. This is an important distinction in being able to determine if metal-deficient SOD1 species are involved in ALS. An SOD1 dimer has 9 different metal-binding combinations distinguishable by mass. The intrasubunit disulfide bond further complicates matters, as it can be in an oxidized or reduced state, altering the mass of the protein by two Da. Indeed several groups have reported that as much as 20-30% of the SOD1 in the ALS model animals is reduced [38
]. Combining these two phenomena, the SOD1 dimer has 27distinctcombinations of metal-binding and disulfide states, which would yield a complex and hard-to-interpret spectrum. An advantage of the described method is that the dimer dissociates in the course of the assayand only monomer is detected (). The SOD1 monomer has only eight distinct metal-binding/disulfide states, with the resulting spectrum being significantly easier to interpret, facilitating analysis of the metal-binding status of individual subunit populations.
A second advantage is that the method is highly adaptable, allowing monitoring of other proteins and post-translational modifications. We have used the method to measure recombinant proteins in solution such as CCS, hemoglobin, and di-iron mono-oxygenases. Other proteins were also observed when measuring SOD1 from tissue, such as ubiquitin and acyl-CoA-binding protein which were tentatively identified by their mass (). The endogenous rat SOD1 was also observable and demonstrates the enormous overexpression of human G93A SOD1 in transgenic rat tissue (). Taking advantage of other materials available for ZipTips (C18, etc.) could further extend the method to additional proteins. Modifying the solvent composition and the ionization parameters, such as the ionization voltage and capillary temperature, allows the method to be optimized for other proteins and post-translational modifications, possibly including small-molecule binding, nitration, metals, andphosphorylation. Furthermore, these modifications can be monitored in a dynamic fashion, as both modified and unmodified populations of a protein are observed at the same time.
The main limitation to our assay is the inability to distinguish between copper-containing, zinc-deficient SOD1 and zinc-containing, copper deficient SOD1. The isotopically broad mass spectrometer signature of a protein is wider than the mass difference between zinc and copper, and thus we cannot differentiate between the two (). However, we find a significant one-metal SOD1 peak that is substantially larger in the ventral gray matter, which provides additional evidence that non-natively metal-bound SOD1 species are involved in the progression of the disease (). Further work that is now ongoing will be needed to determine the composition of the one metal peak and elucidate whether either species has a role in the disease.