New Technique Boosts By Four Times The Size Of A Protein That Researchers Can An

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New Technique Boosts By Four Times The Size Of A Protein That

Researchers Can Analyze

http://www.medicalnewstoday.com/medicalnews.php?newsid=53523

Imagine you had to break a secret code, but you could see only part

of the message. That's the kind of frustration researchers face when

trying to identify proteins and characterize how those proteins are

modified in cells by biological processes.

But now, Cornell researchers have extended a powerful technique to

increase by fourfold the size of a protein that can be analyzed, to

those containing more than 2,000 amino acids, up from about 500.

Called a " top-down " approach, the technique uses a mass spectrometer,

which measures the masses of ions or charged particles. Researchers

break up the protein into pieces and weigh both the masses of the

whole protein and of the individual pieces. By matching the weight of

the whole protein and its pieces with those of known protein

sequences in a database, they can identify the protein. Any

differences in mass with known proteins can help researchers also

find where and how proteins have been modified in cells.

For example, if a section of a protein has an increased weight of 16,

researchers can tell that the protein has been oxidized within that

section, which means that an oxygen atom (with an atomic weight of

16) was added.

" When you isolate a protein from a mixture, your first problem is to

know which one it is, " said Fred McLafferty, Cornell's J.W.

Debye Professor Emeritus of Chemistry and Chemical Biology and senior

author of the paper published in the Oct. 6 issue of the journal

Science. " Mass spectrometry characterizes a protein by measuring the

masses produced from it. "

The new technique provides a far more efficient way to break down

proteins inside the mass spectrometer -- sprayed, heated and then

bashed with gas molecules -- to obtain their pieces for the mass

analysis. They are sprayed in an electric field where they pick up

charges and are vaporized as they pass through a heated capillary.

Then they are hit by air molecules in low-energy collisions to keep

the protein from folding up; then they are slammed with air molecules

in high-energy collisions to dissociate the protein. Next, the mass

spectrometer weighs any surviving protein and its pieces

simultaneously.

The top-down approach rivals the commonly used " bottom-up " approach,

in which proteins are first broken down, or digested, enzymatically

into smaller units of five to 20 amino acids. These pieces are then

introduced into the mass spectrometer where researchers can match

masses of those pieces to known sequences and identify the proteins.

However, this approach reveals less information concerning

modifications to proteins. Such important cellular processes as

oxidation and acetylation of proteins add chemical groups and alter

how a protein functions, such as modifying an enzymatic pathway. The

bottom-up approach is limited because the samples seldom have masses

that represent all the pieces of the protein, and masses can usually

be matched to several combinations of pieces and modifications.

Therefore, the method rarely locates all modifications in newly

isolated proteins.

" Each approach has different strengths and weaknesses, " said Mi Jin,

a postdoctoral associate in chemistry and chemical biology and one of

the paper's lead authors with fellow graduate student Xuemei Han. Jin

said that the bottom-up approach is often used for larger scale

studies of proteins because it can identify a large number of

proteins from a sample, but it does not provide a complete picture of

each protein. The top-down approach, on the other hand, measures the

whole protein and thus provides more confident identifications; it is

also better at revealing modifications and mutations, where there

might be a mistake or addition in the sequence. The new approach can

also provide sequence information on a protein from scratch when it

is not present in any database.

The study was funded by National Institutes of Health.